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[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data.", "We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host.", "In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) .", "Text: Bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and SARS coronavirus (Calisher et al., 2006; Wang and Anderson, 2019) . In most non-Chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (Nicholls et al., 2003; Hooper et al., 2001; Mahanty and Bray, 2004) . Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) .", "Bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (Schountz et al., 2017) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (Plowright et al., 2016) . Recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (Brook and Dobson, 2015) . Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) .", "Bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (Ng et al., 2015; Takadate et al., 2020) and constitutive expression of the antiviral cytokine, IFN-a, for others (Zhou et al., 2016) . Typically, the presence of viral RNA or DNA in the cytoplasm of mammalian cells will induce secretion of type I interferon proteins (IFN-a and IFN-b), which promote expression and translation of interferon-stimulated genes (ISGs) in neighboring cells and render them effectively antiviral (Stetson and Medzhitov, 2006) . In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) .", "In some bat cells, the transcriptomic blueprints for this IFN response are expressed constitutively, even in the absence of stimulation by viral RNA or DNA (Zhou et al., 2016) . In non-flying mammals, constitutive IFN expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (Zhang et al., 2013; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018) that may have evolved to mitigate metabolic damage induced during flight (Kacprzyk et al., 2017) . The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved.", "The extent to which constitutive IFN-a expression signifies constitutive antiviral defense in the form of functional IFN-a protein remains unresolved. In bat cells constitutively expressing IFN-a, some protein-stimulated, downstream ISGs appear to be also constitutively expressed, but additional ISG induction is nonetheless possible following viral challenge and stimulation of IFN-b (Zhou et al., 2016; Xie et al., 2018) . Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated.", "Despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. The field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with HIV, who appeared to produce and clear virus at equivalent rates (Nowak and May, 2000; Ho et al., 1995) . Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms.", "Models of simple target cell depletion, in which viral load is dictated by a bottom-eLife digest Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check.", "Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation.", "In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves.", "However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory.", "have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the Egyptian fruit bat -in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al.", "The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans.", "The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not.", "This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans.", "Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.", "In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for HIV (Perelson, 2002) but have since been applied to other chronic infections, including hepatitis-C virus (Neumann et al., 1998) , hepatitis-B virus (Nowak et al., 1996) and cytomegalovirus (Emery et al., 1999) . Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) .", "Recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza A and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (Baccam et al., 2006; Pawelek et al., 2012; Saenz et al., 2010; Morris et al., 2018) . To investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity.", "We evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. Finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data.", "We hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. We further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture.", "We first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. We conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [1] Vero (African green monkey) cells, which are IFN-defective and thus limited in antiviral capacity (Desmyter et al., 1968) ; [2] RoNi/7.1 (Rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (Kuzmin et al., 2017; Arnold et al., 2018; Biesold et al., 2011; Pavlovich et al., 2018) ; and [3] PaKiT01 (Pteropus alecto) cells which constitutively express IFN-a (Zhou et al., 2016; Crameri et al., 2009) . To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) .", "To intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with GFP-tagged, replication-competent vesicular stomatitis Indiana viruses: rVSV-G, rVSV-EBOV, and rVSV-MARV, which have been previously described (Miller et al., 2012; Wong et al., 2010) . Two of these viruses, rVSV-EBOV and rVSV-MARV, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, Ebola (EBOV) and Marburg (MARV), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a.", "Previous work in this lab has demonstrated incompatibilities in the NPC1 filovirus receptor which render PaKiT01 cells refractory to infection with rVSV-MARV (Ng and Chandrab, 2018, Unpublished results) , making them structurally antiviral, over and above their constitutive expression of IFN-a. All three cell lines were challenged with all three viruses at two multiplicities of infection (MOI): 0.001 and 0.0001. Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1).", "Between 18 and 39 trials were run at each cell-virus-MOI combination, excepting rVSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which only eight trials were run (see Materials and methods; Figure 1 -figure supplements 1-3, Supplementary file 1). Because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (Howat et al., 2006) , we were able to track the spread of GFP-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods).", "For each infection trial, we monitored and re-imaged plates for up to 200 hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see Materials and methods). We used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( Figure 1; Figure 1 -figure supplements 1-5). All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs.", "All three recombinant vesicular stomatitis viruses (rVSV-G, rVSV-EBOV, and rVSV-MARV) infected Vero, RoNi/7.1, and PaKiT01 tissue cultures at both focal MOIs. Post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV.", "Epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rVSV-EBOV and rVSV-MARV. Monolayer destruction was avoided in the case of rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells: in the former, persistent viral infection was maintained throughout the 200 hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) .", "We assumed this pattern to be the result of immune-mediated epidemic extinction (Figure 1) . Patterns from MOI = 0.001 were largely recapitulated at MOI = 0.0001, though at somewhat reduced total proportions (Figure 1-figure supplement 5 ). A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ).", "A theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells We next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( Figure 1 ). The compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (S), antiviral (A), exposed (E), infectious (I), and dead (D). We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication.", "We modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. Antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of ISGs in tissues adjacent to infection (Stetson and Medzhitov, 2006) . Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system.", "Because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. In systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (\") additionally scaled with the global proportion of susceptible cells in the model. Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) .", "Compared with virus, IFN particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of IFN signaling in plaque assay (Howat et al., 2006) . To best represent our empirical monolayer system, we expressed our state variables as proportions (P S , P A , P E , P I , and P D ), under assumptions of frequency-dependent transmission in a wellmixed population (Keeling and Rohani, 2008) , though note that the inclusion of P D (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium.", "This resulted in the following system of ordinary differential equations:\n\nWe defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. By contrast, we allowed the extent of constitutive immunity to vary across the parameter range of \" > 0, defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. In fitting this model to tissue culture data, we independently estimated both r and \"; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e.", "Since the extent to which constitutively-expressed IFN-a is constitutively translated into functional protein is not yet known for bat hosts (Zhou et al., 2016) , this approach permitted our tissue culture data to drive modeling inference: even in PaKiT01 cell lines known to constitutively express IFN-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of \": For the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at 0. The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) .", "The small spatial scale and short time course (max 200 hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (Radke et al., 1974; Rasmussen and Farley, 1975; Samuel and Knutson, 1982) . Before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1.", "From our within-host model system (Equation 1-5), we derived the following expression for R 0 , the pathogen basic reproduction number (Supplementary file 2):\n\nPathogens can invade a host tissue culture when R 0 >1. Rapid rates of constitutive antiviral acquisition (\") will drive R 0 <1: tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with.", "Since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for R 0 ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. In cases of fully induced or absent immunity (\" ¼ 0), the R 0 equation thus reduces to a form typical of the classic SEIR model:\n\nAt equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( Figure 2) . Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ).", "Respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( Figure 1 ). Theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. Endemic equilibria resulting from cellular regeneration (i.e.", "Endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for HIV (Coffin, 1995) and in vitro for herpesvirus plaque assays (Howat et al., 2006) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade.", "Bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (Branch point) values for b, below which the pathogen was unable to invade. We found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles.", "b high enough to drive pathogen-induced extinction), but high b values resulted in Hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. Since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ).", "Under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (\") rates of antiviral acquisition, Hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( Figure 2 ). Consistent with our derivation for R 0 , we found that the Branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of \" that characterize highly constitutive immunity ( Figure 3) . We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity.", "We next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. In general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (Table 1 Ironically, the induced immune model offered a slightly better fit than the constitutive to rVSV-MARV infections on the PaKiT01 cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion.", "Because constitutive immune assumptions can prohibit pathogen invasion (R 0 <1), model fits to this time series under constitutive assumptions were handicapped by overestimations of \", which prohibited pathogen invasion. Only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01.", "In all panel (A) plots, the rate of induced immune antiviral acquisition (r) was fixed at 0.01. Panel (B) depicts dynamics under variably induced immunity, ranging from absent (left: r=0) to high (right: r=1). In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "In all panel (B) plots, the rate of constitutive antiviral acquisition (\") was fixed at 0.0001 Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4).", "In fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + \") ( Table 1 ; Supplementary file 4). Under absent immune assumptions, r and \" were fixed at 0 while b was estimated; under induced immune assumptions, \" was fixed at 0 while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, \", and b) were simultaneously estimated for each cell-virus combination. Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\").", "Best fit parameter estimates for MOI=0.001 data are visualized in conjunction with br and b -\" bifurcations in (r) and (B) the constitutive immunity rate of antiviral acquisition (\"). Panels show variation in the extent of immunity, from absent (left) to high (right). Branch point curves are represented as solid lines and Hopf curves as dashed lines.", "Branch point curves are represented as solid lines and Hopf curves as dashed lines. White space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0.", "Other parameter values for equilibrium analysis were fixed at: b = .025, m = .001, s = 1/6, a = 1/6, c = 0. Special points from bifurcations analyses are listed in Supplementary file 3. space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) .", "In contrast to Vero cells, the induced immunity model offered the best fit to all RoNi/7.1 data, consistent with reported patterns in the literature and our own validation by qPCR ( Table 1; Arnold et al., 2018; Kuzmin et al., 2017; Biesold et al., 2011; Pavlovich et al., 2018) . As in Vero cell trials, we estimated highest b values for rVSV-G infections on RoNi/7.1 cell lines but here recovered higher b estimates for rVSV-MARV than for rVSV-EBOV. This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV.", "This reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rVSV-EBOV versus rVSV-MARV. In general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, \", or both) correlated with higher transmission rates (b). When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) .", "When offset by r, b values estimated for RoNi/7.1 infections maintained the same amplitude as those estimated for immune-absent Vero cell lines but caused gentler epidemics and reduced cellular mortality (Figure 1) . RoNi/7.1 parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (Figure 4) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4).", "Finally, rVSV-G and rVSV-EBOV trials on PaKiT01 cells were best fit by models assuming constitutive immunity, while rVSV-MARV infections on PaKiT01 were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in AIC comparisons because one fewer parameter was estimated (Figure 1-figure supplements 4-5; Supplementary file 4). For all virus infections, PaKiT01 cell lines yielded b estimates a full order of magnitude higher than Vero or RoNi/7.1 cells, with each b balanced by an immune response (either r, or r combined with \") also an order of magnitude higher than that recovered for the other cell lines ( Figure 4 ; Table 1 ). As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model.", "As in RoNi/7.1 cells, PaKiT01 parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. Because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rVSV-MARV infections on PaKiT01 cell lines consistently localized at or below the Branch point threshold for virus invasion (R 0 ¼ 1). During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data.", "During model fitting for optimization of \", any parameter tests of \" values producing R 0 <1 resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. In all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from \" prohibited virus from invading at all. The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled.", "The induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. In order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-MOI combination-based on the following equation:\n\nwhere P E was calculated from the initial infectious dose (MOI) of each infection experiment and P S was estimated at disease-free equilibrium:\n\nBecause and \" both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) .", "Indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rVSV-MARV infection on PaKiT01 cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (Supplementary file 4) . In reality, we know that NPC1 receptor incompatibilities make PaKiT01 cell lines constitutively refractory to rVSV-MARV infection (Ng and Chandrab, 2018, Unpublished results) and that PaKiT01 cells also constitutively express the antiviral cytokine, IFN-a. Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein.", "Model fitting results suggest that this constitutive expression of IFN-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional IFN-a protein. Nonetheless, as hypothesized, PaKiT01 cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( Table 1 ; Supplementary file 4). RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4).", "RoNi/7.1 cells displayed the second-most-pronounced signature of immunity, followed by Vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( Table 1 ; Supplementary file 4). Using fitted parameters for b and \", we additionally calculated R 0 , the basic reproduction number for the virus, for each cell line-virus-MOI combination ( Table 1 ; Supplementary file 4). We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low.", "We found that R 0 was essentially unchanged across differing immune assumptions for RoNi/7.1 and Vero cells, for which the initial antiviral rate was low. In the case of PaKiT01 cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, R 0 ) which still produced the same epidemic curve that resulted from the much lower estimates for b and R 0 paired with absent immunity. These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates.", "These findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. Total monolayer destruction occurred in all cell-virus combinations excepting rVSV-EBOV infections on RoNi/7.1 cells and rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells. Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) .", "Monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where R-effective (the product of R 0 and the proportion susceptible) was reduced below one ( Figure 5) . For rVSV-EBOV infections on RoNi/7.1, induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined.", "In rVSV-EBOV and rVSV-MARV infections on PaKiT01 cells, this antiviral protection halted the epidemic ( Figure 5 ; R-effective <1) before susceptibles fully declined. In the case of rVSV-EBOV on PaKiT01, the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) .", "Importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rVSV-EBOV infections on PaKiT01 cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of IFN-mediated viral dynamics in tissue culture (Howat et al., 2006) . In our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model.", "Finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. In spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and \" derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see Materials and methods; Videos 1-3; Figure 5-figure supplement 3; Supplementary file 5; Webb et al., 2007) . In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer.", "In immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. Eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (Videos 1-3; Figure 5-figure supplement 3 ). Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens.", "Bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. Though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (Zhou et al., 2016; Ahn et al., 2019; Xie et al., 2018; Pavlovich et al., 2018; Zhang et al., 2013) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics.", "We used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. Critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture.", "These results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. Additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts.", "Our findings suggest that viruses evolved in bat reservoirs possessing enhanced IFN capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. Such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions.", "To achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. We considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for \", the constitutive rate of antiviral acquisition. In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \".", "In deriving the equation for R 0 , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (R 0 >1), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, \". Constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates.", "Once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. Regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature.", "Fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. Simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from IFN-deficient Vero cells, while models assuming induced immune processes most accurately reproduced trials derived from RoNi/7.1 (Rousettus aegyptiacus) cells, which possess a standard virusinduced IFN-response. In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) .", "In most cases, models assuming constitutive immune processes best recreated virus epidemics produced on PaKiT01 (Pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . Model support for induced immune assumptions in fits to rVSV-MARV infections on PaKiT01 cells suggests that the constitutive IFN-a expression characteristic of P. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series.", "Results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. As previously demonstrated in within-host models for HIV (Coffin, 1995; Perelson et al., 1996; Nowak et al., 1995; Bonhoeffer et al., 1997; Ho et al., 1995) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line.", "Critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral PaKiT01 P. alecto cell line. These findings indicate that enhanced IFN-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines.", "We nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. Future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference.", "antiviral cells) to enhance epidemiological inference. The continued recurrence of Ebola epidemics across central Africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) .", "The past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (Brook and Dobson, 2015; Xie et al., 2018; Zhang et al., 2013; Ahn et al., 2019; Zhou et al., 2016; Ng et al., 2015; Pavlovich et al., 2018) . Nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) .", "Endemic maintenance of infection is a defining characteristic of a pathogen reservoir (Haydon et al., 2002) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (Peel et al., 2012) . Researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (Plowright et al., 2016) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (Peel et al., 2018; Brook et al., 2019) . We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes.", "We present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. Viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) .", "All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009) . The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009) . Vero cells were obtained from ATCC.", "Vero cells were obtained from ATCC. Monolayers of each cell line were grown to 90% confluency (~9Â10 5 cells) in 6-well plates. Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies).", "Cells were maintained in a humidified 37˚C, 5% CO 2 incubator and cultured in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) .", "Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-b deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979) . RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011) , while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-a (Zhou et al., 2016) . This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below.", "This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.", "Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells. Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012) . Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor.", "Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines. To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection.", "To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH 4 Cl (20 mM) was added to infected cell cultures at 1-2 hr postinfection to block viral spread, and individual eGFP-positive cells were manually counted at 12-14 hr post-infection. Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016 ) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection.", "We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-a and IFN-b genes across a longitudinal time series of infection. Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001-with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time.", "All experiments were run in duplicate on 6well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI. For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C.", "For these gene expression time series, four 6-well plates for each cell line-virus combination were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously).", "Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer's instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions.", "Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer's instructions. cDNA was then stored at 4˚C and as a frozen stock at À20˚C to await qPCR. We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) .", "We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-a and IFNb, and the housekeeping gene, b-Actin, using primers previously reported in the literature (Supplementary file 6) . For qPCR, 2 ml of each cDNA sample was incubated with 7 ml of deionized water, 1 ml of 5 UM forward/reverse primer mix and 10 ml of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 C for 2 min followed by 40 cycles of: denaturation at 95˚C (5 s), annealing at 58˚C (15 s), and extension at 72˚C (10 s). We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6.", "We report simple d-Ct values for each run, with raw Ct of the target gene of interest (IFN-a or IFN-b) subtracted from raw Ct of the b-Actin housekeeping gene in Figure 1 -figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the d-d-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-a in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells. After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV).", "After being grown to~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k!", "In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: P k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: 1 À e Àm . We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model.", "We assumed that a~90% confluent culture at each trial's origin was comprised of~9x10 5 cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively,~81 or 810 cells, representing the state variable 'E' in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate.", "Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2-3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37˚C.", "Cells were incubated with virus for one hour at 37˚C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/Lglutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37˚C, 5% CO 2 environment. After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation).", "After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6-9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64-or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image.", "Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1-figure supplement 1) . Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.", "Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining. In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform.", "Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.", "One color channel was permitted such that images produced showed live nuclei in white and dead cells in black. Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015) . As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e.", "As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e.", "Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFPexpressing images and used Hoechst stain images as a post hoc visual check on our fit only ( Figure 5 ; Figure 5 -figure supplements 1-2). Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7.", "Images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for Hoechst-stained images, 'live' vs. 'dead') form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e.", "Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool.", "As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-MOI combinations (Figure 1 \n\nTo derive the expression for R 0 , the basic pathogen reproductive number in vitro, we used Next Generation Matrix (NGM) techniques (Diekmann et al., 1990; Heffernan et al., 2005) , employing Wolfram Mathematica (version 11.2) as an analytical tool. R 0 describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when R 0 >1 (Supplementary file 2). We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3).", "We then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using MatCont (version 2.2) (Dhooge et al., 2008) for Matlab (version R2018a) (Supplementary file 3). The birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. As such, we fixed b at.", "As such, we fixed b at. 025 and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via Hoechst staining of control wells for each of the three cell lines (Figure 1-figure supplement 7) . This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) .", "This yielded a natural mortality rate, m, corresponding to a lifespan of approximately 121, 191, and 84 hours, respectively, for Vero, RoNi/7.1, and PaKiT01 cell lines (Figure 1-figure supplement 7) . We then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging 6 to 9.5 hours). We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials.", "We fixed a, the infection-induced mortality rate, at 1/6, an accepted standard for general viral kinetics (Howat et al., 2006) , and held c, the rate of antiviral cell regression to susceptible status, at 0 for the timespan (<200 hours) of the experimental cell line infection trials. We estimated cell line-virus-MOI-specific values for b, r, and \" by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (Figure 1-figure supplements 2-3) . Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep.", "Fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-MOI-specific infectious proportion of the data at each timestep. We optimized parameters for MOI = 0.001 and 0.0001 simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and \" across the two time series. We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001).", "We used the differential equation solver lsoda() in the R package deSolve (Soetaert et al., 2010) to obtain numerical solutions for the mean field model and carried out minimization using the 'Nelder-Mead' algorithm of the optim() function in base R. All model fits were conducted using consistent starting guesses for the parameters, b (b = 3), and where applicable, r (r = 0.001) and \" (\" = 0.001). In the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model.", "All eighteen cell line-virus-MOI combinations of data were fit by an immune absent (\" = r = 0) version of the theoretical model and, subsequently, an induced immunity (\" = 0; r >0) and constitutive immunity (\" >0; r >0) version of the model. Finally, we compared fits across each cell line-virus-MOI combination via AIC. In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions.", "In calculating AIC, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and \") for constitutive immune assumptions. The sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.", "All fitting and model comparison scripts are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807. Finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) .", "We constructed our spatial model in C++ implemented in R using the packages Rcpp and RcppArmadillo (Eddelbuettel and Francois, 2011; Eddelbuettel and Sanderson, 2017) . Following Nagai and Honda (2001) and Howat et al. (2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions.", "(2006) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. At the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model.", "Parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. Transitions involving the induced (r) and constitutive (\") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep.", "As such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and \" by the global proportion of, respectively, exposed and susceptible cells at a given timestep. In contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell.", "The birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. To compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (Webb et al., 2007) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (Supplementary file 4) . We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible.", "We derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small (7.5%) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. We justify these increases and derive their origins further in Supplementary file 5. We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in .", "We simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of 10,000 cells and compared model output with data in . Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. All images and code used in this study have been made available for download at the following Figshare" ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\n\nJournal Pre-proof\nZixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin\nSong\nPII: S1546-1440(20)30285-4\nDOI: \nReference: JACR 5139\nTo appear in: Journal of the American College of Radiology\nReceived Date: 24 February 2020\nRevised Date: 13 March 2020\nAccepted Date: 15 March 2020\nPlease cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against\nCoronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology\nDepartment, Journal of the American College of Radiology (2020), doi: \nj.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition\nof a cover page and metadata, and formatting for readability, but it is not yet the definitive version of\nrecord. This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article.", "This version will undergo additional copyediting, typesetting and review before it is published\nin its final form, but we are providing this version to give early visibility of the article. Please note that,\nduring the production process, errors may be discovered which could affect the content, and all legal\ndisclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author.", "© 2020 Published by Elsevier Inc. on behalf of American College of Radiology\nThe Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management\nand Infection Control in a Radiology Department\nZixing Huang*, Shuang Zhao*, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng,\nBin Song\nDepartment of Radiology, West China Hospital, Sichuan University, Chengdu, China\n*Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD\nAddress: Department of Radiology, West China Hospital, Sichuan University. No.", "No. 37, GUOXUE Alley, Chengdu, 610041, China\nTel. : (+86)28 85423680, Fax: (+86)28 85582944\nEmail: [email protected]. Authors’ contributions\nZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript.", "ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT)\nand was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the\nmeasures.", "WXC: The member of the EMICT and was involved in the formulation of the\nmeasures. LHZ: The member of the EMICT and was involved in the formulation of the\nmeasures. LPD: The member of the EMICT and was involved in the formulation of the\nmeasures.", "LPD: The member of the EMICT and was involved in the formulation of the\nmeasures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.", "The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the\nauthors take complete responsibility for the integrity of the data and the accuracy of\nthe data analysis\n1\nThe Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency\nManagement and Infection Control in a Radiology Department\nAbstract\nObjective: To describe the strategy and the emergency management and infection control\nprocedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams.", "Methods: We set up emergency management and sensing control teams. The team formulated\nvarious measures: reconfiguration of the radiology department, personal protection and training\nof staff, examination procedures for patients suspected of or confirmed with COVID-19 as well\nas patients without an exposure history or symptoms. Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit.", "Those with suspected or confirmed\nCOVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with\nCOVID-19 underwent fever-CT examinations. Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340.", "Including initial examinations and\nreexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our\nprecautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity.", "Conclusion: Strategic planning and adequate protections can help protect patients and staff\nagainst a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control\n\n2\nIntroduction\nThe whole world has been closely focusing on an outbreak of respiratory disease caused by a\nnovel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that\ncontinues to spread. On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19).", "On February 11, 2020, the World Health Organization (WHO) named the\ndisease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports\nof 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized.", "There remain 14,831\nconfirmed cases (including 4,257 in serious condition) and 253 suspected cases still\nhospitalized. To date, 677,243 people have been identified as having had close contact with\ninfected patients of whom13,701 are under medical observation [1]. Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2].", "Outside China, 44,067\nlaboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas\naccording to the WHO [2]. COVID-19 poses significant threats to international health. Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes.", "Like the\nflu, COVID-19 is thought to spread mainly from person-to-person between people who are in\nclose contact with one another through respiratory droplets produced when an infected person\ncoughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at\nhigh risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3].", "In China, healthcare workers account for 1,716 confirmed\ncases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing\nCOVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines\nfrom the NHC [4]. Suspected patients having the virus may undergo chest CT.", "Suspected patients having the virus may undergo chest CT. Isolation and\nbarrier procedures are necessary to protect both the department staff and other patients in the\nhospital. Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool.", "Note should be made that due to overlap of imaging findings with other respiratory\n3\ndiseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary\ninvolvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province.", "Our hospital is a national regional medical center with 4,300 beds and a tertiary referral\ncenter in Sichuan province. The initial response started on January 21, 2020, after transmission\nof COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020.", "The first suspected\ncase of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan\nprovincial government immediately launched the first-level response to major public health\nemergencies. On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19.", "On the same day, our hospital was designated to care for Sichuan province\npatients with COVID-19. This article describes the emergency management procedure of our radiology department\nfor situations involving severe infectious diseases, such as COVID-19, and the\ninfection-protection experience of the department staff. Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward.", "Methods\nThe hospital provided personal protective equipment (medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected\nthree medical tents (fever tents) for screening of fever cases in the parking lot of the emergency\ndepartment, planned an examination route and examination area for patients suspected of\nharboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the\ncolloquial term used to designate suspected COVID-19 based on symptoms such as a fever or\nwith an epidemiological history of a potential exposure as well as those with confirmed\nCOVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria.", "Further, during outbreak, emergency and outpatient patients\n4\nwithout fever were asked for information such as epidemiological history and sent to fever tents\nas long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members\n(trained technologists, nurses, engineers, and support staff). The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS).", "The equipment of the radiology\ndepartment includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital\nsubtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems\n(including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture\narchiving and communication systems (PACS). Most of the equipment is distributed among\nfour buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor.", "4 CT scanners, 4 MR scanners, 1 DR are located on\nthe first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second\nfloor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner\nare located in the sixth inpatient building.", "1 CT and 1 MR scanner\nare located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located\nin the technical building. The rest of the equipment is located in the seventh inpatient building\nin the branch campus.", "The rest of the equipment is located in the seventh inpatient building\nin the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to\nhandle cases of COVID-19. Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19.", "Fever tents were set up by the emergency department in the\nemergency department parking lot to separate normal emergency patients from patients with\nsymptoms or exposure history suspicious of COVID-19. We established separate means of\naccess between fever tents and between the fever examination area of the radiology department\nto avoid cross-contamination. The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital.", "The emergency management and infection control measures, as described below and\nimplemented in the radiology department during the outbreak, have been approved by the\n5\ninfection control committee of hospital. These measures are in accordance with relevant laws\nand regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT.", "Radiology Emergency Management and Infection Control Team (EMICT)\nThe radiology department director chaired the EMICT. Its members include the deputy\ndirector, chief technologist, head nurse, equipment engineer supervisor, and infection control\nnurse of the radiology department. Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally.", "Team responsibilities included (1) coordination between the\nhospital’s management and planning of infection control and radiology departments; (2)\ncollection of the most up-to-date protection-related information to educate and train staff in the\ndepartment; (3) reallocation of staff according to the actual situation; (4) establishment of the\nCT procedures for patients with COVID-19; and (5) establishment of an emergency\nmanagement plan for the radiology department to ensure that the department would run\nnormally. Suspected patients\nThe suspected patients were identified according to the Diagnosis and Treatment Program of\nthe Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1).", "Reconfiguration of the radiology department\nThe radiology department was divided into four areas [6]: contaminated, semicontaminated,\nbuffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and\nincludes the fever accessway, the CT examination room, and the DR examination room for\n6\nconfirmed and suspected cases. One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19.", "One CT scanner and one DR system closest to the emergency\ndepartment are designated the fever-CT and fever-DR to examine patients with suspected and\nconfirmed COVID-19. There is a separate dedicated access between the contaminated area and\nthe fever screening tents. The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas.", "The semicontaminated area includes the fever-CT control room,\nfever-DR control room, and other patient examination access areas. The buffer zone includes\naccess areas for medical personnel and a dressing area for technologists. The clean area\nincludes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades.", "The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles.", "Personal protection and training of staff\nFor providing care for patients with confirmed and suspected COVID-19, all hospital staff\nare required to wear complete personal protective equipment [7]: medical protective clothing,\nsurgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the\nequipment must be performed in accordance with the procedures and under the supervision of\nthe infection control nurse. Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels.", "Because staff members working in the contaminated area are under much situational\npressure, periodically taking time off could lower their physical and mental stress levels. The\ntechnologists on fever-CT duty shifts are provided a break once a week for four hours. In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19.", "In\naddition, the health of staff in the contaminated area must be monitored closely for the\nsymptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University.", "7\nThe EMICT formulates and continually updates guidelines and educates all staff for West\nChina Hospital of Sichuan University. The EMICT training for staff is mainly involves\ndocuments regarding infection control and CT findings of COVID-19 and maintains an EMICT\nWeChat group for West China Hospital of Sichuan University. WeChat is the most widely used\nsocial media app in China.", "WeChat is the most widely used\nsocial media app in China. The EMICT releases the latest national and hospital-based\ninformation regarding COVID-19, guidance documents, and other notices from the hospital\nand radiology department in the WeChat group on a daily basis. Staff can also report to the\nEMICT in the WeChat group any time.", "Staff can also report to the\nEMICT in the WeChat group any time. Protocols for each modality and infection control\ninstructions are posted on the walls in all examination rooms. The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty.", "The EMICT periodically reminds\nstaff to undertake personal measures to reduce infection, such as wearing masks at all instances\nin the radiology department and N95 masks if working in the contaminated area; not touching\nthe mask and the eyes; practicing hand hygiene; facing away from colleagues when eating,\ndrinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and\ntechnologists on typical CT findings of COVID-19 infection using materials developed in\nWuhan, the epicenter of the outbreak in China. CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19.", "CT examination procedures\nThere are two sets of procedures for CT examination: the fever-CT procedure and routine CT\nprocedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2)\n8\nBefore the fever-CT technologist operates the equipment, he or she should wear personal\nprotective equipment according to three-level protection standard [8]. Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance.", "Before the CT\nexamination of patients with suspected and confirmed COVID-19 begins, the fever tent or\nisolation ward notifies the radiologist in advance. The fever-CT technologist checks the\nequipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area.", "The patient enters the fever-CT waiting area through the fever access area. If the patient\ncan get onto and off the examination table by themselves, the patient is allowed to do so. If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist.", "If the\npatient cannot get onto or off the examination table independently, the person accompanying\nthe patient assists the patient, rather than the technologist. The technologist checks the patient\ninformation and, using an intercom system in the examination room, asks the patient to remove\nany metal ornaments on the neck and chest. Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination.", "Also, by intercom, the technologist trains the\npatient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the\noriginal images are reconstructed as 1 mm-thick layers.", "After scanning, the\noriginal images are reconstructed as 1 mm-thick layers. The technologist browses the images to\nensure that their quality meets the diagnostic requirements and then guides the patient to leave\nthrough the fever access area. The disposable sheets for patient examination are changed after\neach patient. The equipment is disinfected according to the procedure below.", "The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and\nother personal protection as established by the EMICT. The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons.", "The CT procedure for regular patients (figure.3)\n9\nSome patients with COVID-19 have no symptoms, and they may call at the general clinic for\nother reasons. The following CT procedure is applicable under these circumstances:\nWhen the patient makes an appointment for examination, the staff asks the patient about\ntheir epidemiological history, symptoms, and signs. If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening.", "If suspected criteria are met, the patient\nwill be sent to the fever tent for further screening. When a patient presents to the radiology\ndepartment entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation.", "If the temperature is higher than 37.2 , ℃\nthe patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the\nnon-contaminated CT scanners. The technologists assigned to these scanners wear surgical\nmasks.", "The technologists assigned to these scanners wear surgical\nmasks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly.", "After the CT examination, the technologist browses the images quickly. If the CT appearance is\ntypical of lung infection, the technologist immediately reports it to the chest radiologist on duty\nand asks the patient to wait in the CT examination room. If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room.", "If the chest radiologist does not\nsuspect COVID-19 infection, the patient can leave the CT examination room. If the chest\nradiologist does suspect COVID-19 infection, the technologist immediately reports it to the\nEMICT and sends the patient to the fever tent. The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients.", "The floor and equipment in the CT examination\nroom are disinfected according to regulations, and air disinfection is conducted for 30 min\nbefore examining other patients. These CT scanners are considered noncontaminated (not\nfever-CTs) after these sterilization procedures. Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited.", "Fever-DR examination procedure\n10\nThe COVID-19 guideline of the NHC does not recommend chest DR because its ability in\ndiagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide\nbedside examination for critically ill patients. The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below.", "The technologist operating the mobile DR\nwears personal protective equipment according to the three-level protection standard and\nsterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures\nRoutine disinfection procedure [9]\n1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing\ndisinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant.", "2) Equipment disinfection: The equipment in the contaminated area are wiped with\n2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated\narea are wiped with 75% ethanol. The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day.", "The equipment in the buffer area is wiped with\n500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable\ndisinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with\neach other. Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection.", "Polluted area: open the door for ventilation, each time more than 30 minutes,\nonce /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is\ncontinuously used in the unmanned state for 60 minutes, four times a day, remembered to\nclose the inner shielding door when air disinfection. Other ambient air is sprayed with\n1000mg/L chlorine-containing disinfectant and ventilated twice a day\n4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing\ndisinfectant, once /4 hours. 5) When contaminated, disinfect at any time.", "5) When contaminated, disinfect at any time. In case of visible contamination, disposable\nabsorbent materials should be used first to completely remove the pollutants, and then a\ncloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30\nminutes before wiping. 11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10].", "11\nFever-CT disinfection procedures after examination\nIn addition to the above, disinfect the examination bed and ground with chlorinated disinfectant\ncontaining 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination\nIn addition to the above routine disinfection procedure, air disinfection is conducted for 30 min\nbefore examining other patients. Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19.", "Results\nFrom January 21, 2020 when screening for epidemiological history or symptoms\nsuspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals\nand confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT\nexaminations. Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340.", "Including the initial examination and reexamination, the total number of fever\nCT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5\nminutes. As a result of our precautions, none of the staff of the radiology department developed\nsymptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative.", "The fever-CT technologist, with the highest probability\nof exposure, remains PCR negative. Discussion\nIt has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last\nnational spread of severe infectious disease, broke out. Currently, the Chinese people are\npanicking again.", "Currently, the Chinese people are\npanicking again. The speed and extent by which COVID-19 has spread in 2 months are\n12\nunprecedented, beyond those of SARS, and this has been aided by its contagious nature and\nrapid spread via droplets and contact. The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces.", "The droplet mode of transmission means that a person can\nbe infected easily by means of casual contact or even fomites on contaminated environmental\nsurfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients.", "How radiology departments respond to any infectious disease outbreak is determined\nprimarily by the estimated risk of cross-infection to the staff and other patients. Appropriate\nprecautions taken only by staff in direct contact with patients may be adequate when the risk is\nlow. The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high.", "The strongest measures need to be implemented to limit the spread of the disease when the\nrisk is high. With severe infectious diseases such as COVID-19, the highest level of infection\ncontrol measures must be implemented; these include providing adequate standard protective\nequipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10].", "Once a contagious infectious disease has been identified, the EMICT must consider four\nmain areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about\nconfirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions\nto be taken. All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings.", "All typical and atypical imaging features of the disease should be made known to\nall radiologists to assist in recognition of the disease on images and to allow accurate reporting\nof these findings. We have stored images of all probable cases of COVID-19 in the PACS so\nthat these images were readily available for any radiologist to review, and images from\nprevious imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance.", "Collaboration with the radiology departments of other hospitals is very important because\npatients may initially present to different centers, depending on geographic location and travel\n13\ndistance. These patients may be few in number at a single hospital, but if data from patients at\nseveral hospitals are available, a more accurate overall understanding of both imaging features\nand epidemiology can be achieved. Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted.", "Dissemination of this information to all healthcare facilities\nwill also lead to early recognition of the disease, and appropriate isolation measures may be\ninstituted. The Internet and social media apps, especially WeChat, have been used for distribution of\nmedical information, and because the exchange of information regarding infectious disease\noutbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time.", "In fact, within a\nmonth of the outbreak, the hospital that received the most infected patients from the source of\nthe outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which\nwas shared via WeChat and disseminated across the country in a very short time. Subsequently,\nCOVID-19-teaching PowerPoint presentations from various hospitals appeared and were\nquickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity.", "Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19\nbecause of lack of imaging specificity. But when combined with other epidemiological,\nclinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are\nhelpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19.", "In our opinion, the major role of chest CT is to understand the\nextent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we\nadopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been\nwidely used in the screening of early lung cancer. It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19.", "It is well known that many early lung cancers\nare ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for\nCOVID-19. In addition, considering the rapid development of COVID-19, many CT\n14\nexaminations may be conducted in the same individual to monitor disease progress. Low-dose\nscanning can reduce the radiation damage to patients.", "Low-dose\nscanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary\npersonnel and other patients, it remains limited as follows. Sichuan province is not the center of\nthe epidemic.", "Sichuan province is not the center of\nthe epidemic. The number of patients with COVID-19 whom we have treated has not been\nhigh, and most cases are from other provinces of China. However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19.", "However, we believe that our\nexperience in management, the reconfiguration of our radiology department, and the workflow\nchanges implemented in the current COVID-19 situation are useful for other radiology\ndepartments that must prepare for dealing with patients with COVID-19. While no radiology\npersonnel developed symptoms suspicious for or were confirmed as having COVID-19, there\nmay be asymptomatic personnel. REFERENCES\n1.", "REFERENCES\n1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing\non novel coronavirus cases in China. Retrieved from\n Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9.", "Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from\n\n2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in\nepidemic control on Feb 15. Retrieved from Accessed\nMarch 11, 2020. 15\n4.", "Retrieved from Accessed\nMarch 11, 2020. 15\n4. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020\n5.", "Retrieved from\n\nAccessed March 11, 2020\n5. Health Commission of the People’s Republic of China.(2020). The notification of the trial\noperation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from\n\nAccessed March 11, 2020. 6.", "Retrieved from\n\nAccessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens\nisolated operations in hospital. Retrieved from\n Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 8. Health Commission of the People’s Republic of China.(2017).", "Health Commission of the People’s Republic of China.(2017). The guideline for prevention and\ncontrol of hospital acquired infections of airborne pathogens. Retrieved from\n Accessed\nMarch 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital.", "The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for\nsterilization techniques in hospital. Retrieved from\n Accessed March 11, 2020. 11.", "Retrieved from\n Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and\nPhysicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1.", "Accessed March 11,\n2020. 16\nFigure Legends\nFigure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white).", "Diagram of the layout of our radiology department was divided into four areas: contaminated\n(shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas\n(shaded in white). The contaminated area was separated from other areas by barriers. Figure 2.", "The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients.", "Figure 3. Diagram shows CT protocol for regular patients. Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed.", "Abbreviations:\nCOVID-19: coronavirus disease 2019\nCT: computed tomography\nDR: digital radiography\nEMICT: emergency management and infection control team\nNHC: National Health Commission\nPACS: picture archiving and communication system\nSARS: severe acute respiratory syndrome\n\n\n\nSentence Summary\nWith severe infectious diseases such as COVID-19, the highest level of infection control\nmeasures must be implemented, collaboration with the radiology departments of other\nhospitals be needed, and social media be employed. Take-home points\n1. To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT).", "To response to a community infection emergency, a special emergency management team\nneeds to be setup at the departmental level to implement infection containment and\ncontrol procedures that continues to allow the imaging examination and imaging\ndiagnosis of those with suspected infection, and to prevent intra-departmental spreading\nof infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly.", "Infection control measures, such as reconfiguration of department areas, personal\nprotection and anti-infection training of all staff, standardized procedures including\ncontact minimization for chest CT and DR examinations, and timely disinfection of CT\nand DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to\nsuspected cases." ]

[ "This review highlights the main points which emerged from the presentations and discussions at the 3rd isirv-Antiviral Group Conference - advances in clinical management. The conference covered emerging and potentially pandemic influenza viruses and discussed novel/pre-licensure therapeutics and currently approved antivirals and vaccines for the control of influenza. Current data on approved and novel treatments for non-influenza respiratory viruses such as MERS-CoV, respiratory syncytial virus (RSV) and rhinoviruses and the challenges of treating immunocompromised patients with respiratory infections was highlighted.", "Current data on approved and novel treatments for non-influenza respiratory viruses such as MERS-CoV, respiratory syncytial virus (RSV) and rhinoviruses and the challenges of treating immunocompromised patients with respiratory infections was highlighted. Text: Recurrent infections by influenza and other respiratory viruses contribute enormously to the burden of human disease and (emergent) sporadic zoonotic infections, such as by influenza H7N9 and H5N1 and Middle East respiratory syndrome (MERS) coronavirus, pose a constant threat of a new global epidemic. Despite extensive knowledge of the viruses and their interaction with the host, there is little in our armoury of vaccines and therapeutics to combat this perpetual onslaught.", "Despite extensive knowledge of the viruses and their interaction with the host, there is little in our armoury of vaccines and therapeutics to combat this perpetual onslaught. The 3rd isirv Antiviral Group conference on Influenza and Other Respiratory Virus Infections: Advances in Clinical Management, convened in Tokyo, Japan on 4-6 June 2014, attracted 188 clinicians, public health specialists and medical scientists from 34 countries to present their recent research and discuss various aspects of the impact of respiratory viruses in different patient groups/settings and in different regions of the world. The programme 1 focused on the latest advances in the mitigation and clinical management of influenza and other respiratory virus disease, and the successful use of antivirals (and vaccines) against seasonal and pandemic influenza, particularly in Japan, as well as the development/assessment of novel antiviral agents.", "The programme 1 focused on the latest advances in the mitigation and clinical management of influenza and other respiratory virus disease, and the successful use of antivirals (and vaccines) against seasonal and pandemic influenza, particularly in Japan, as well as the development/assessment of novel antiviral agents. This overview highlights some of the main points which emerged from the presentations (both oral and poster) and associated discussion. In recent years, an increasing number of cases of novel animal influenza A viruses infecting humans have been reported.", "In recent years, an increasing number of cases of novel animal influenza A viruses infecting humans have been reported. These include multiple avian influenza A virus subtypes, in particular H5N1 and H7N9, and swine-origin H3N2v. Reasons for this increase include both social factors, for example, increased human populations living in close proximity to animals and increased surveillance and diagnostic testing.", "Reasons for this increase include both social factors, for example, increased human populations living in close proximity to animals and increased surveillance and diagnostic testing. Risk assessment tools have been developed by many authorities around the world (e.g. the European CDC, US CDC, USAID and WHO) to assist in predicting the likelihood that a particular virus will emerge and its associated impact, as well as prioritising the development of candidate human vaccine viruses.", "the European CDC, US CDC, USAID and WHO) to assist in predicting the likelihood that a particular virus will emerge and its associated impact, as well as prioritising the development of candidate human vaccine viruses. 2 These tools allow continual reassessment as new data become available and provide an objective, transparent process with which to make resource allocation and pandemic planning decisions. In February 2013, China detected the first human cases of H7N9 infection in severely ill patients with pneumonia.", "In February 2013, China detected the first human cases of H7N9 infection in severely ill patients with pneumonia. 3 As of May 22, 2014 , there have been 446 confirmed H7N9 cases in China resulting in 163 deaths. 4 The cases have occurred mainly during two waves (weeks [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 2013 and week 40, 2013week 20, 2014) , 4 of which 85% had prior exposure to poultry or contaminated live poultry markets.", "4 The cases have occurred mainly during two waves (weeks [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 2013 and week 40, 2013week 20, 2014) , 4 of which 85% had prior exposure to poultry or contaminated live poultry markets. The median time from poultry exposure to disease onset was 5 days, whereas the median time from illness onset to hospital admission, ARDS development, antiviral therapy and death was 5, 6Á8, 7 and 14 days, respectively. 5 Closure of live poultry markets has markedly reduced the risk of H7N9 infection.", "5 Closure of live poultry markets has markedly reduced the risk of H7N9 infection. Across nine areas in the two most affected provinces in China, modelling analysis estimated that the effectiveness of market closure was 97% (95% CI: 89%, 100%). 6 A retrospective serological study of blood specimens taken in January-May and October-November in 2012 from 1544 subjects who worked in live poultry markets, farms, slaughter houses or kept backyard poultry revealed no evidence of H7N9 infection, 7 indicating widespread population susceptibility and lack of prior circulation of antigenically related viruses.", "6 A retrospective serological study of blood specimens taken in January-May and October-November in 2012 from 1544 subjects who worked in live poultry markets, farms, slaughter houses or kept backyard poultry revealed no evidence of H7N9 infection, 7 indicating widespread population susceptibility and lack of prior circulation of antigenically related viruses. Multiple family clusters have been reported, but no sustained human-to-human transmission, with studies demonstrating a very low detection of virus or specific antibody in close contacts (0Á34% and 0Á2% in the first and second waves, respectively) and healthcare workers of positive cases. 8 In both the first and the second waves, the majority of the patients hospitalised with H7N9 infection were older men (median age, 62 and 58 years, respectively with an overall male/female ratio of 2Á2:1) and the case fatality was similar (32% and 39%, respectively).", "8 In both the first and the second waves, the majority of the patients hospitalised with H7N9 infection were older men (median age, 62 and 58 years, respectively with an overall male/female ratio of 2Á2:1) and the case fatality was similar (32% and 39%, respectively). Pre-existing medical conditions occurred in >60% of these cases. The prominent clinical features on admission were those of a severe influenza syndrome with fever, cough, fatigue and dyspnoea, while the most striking laboratory findings were marked lymphopenia and thrombocytopenia.", "The prominent clinical features on admission were those of a severe influenza syndrome with fever, cough, fatigue and dyspnoea, while the most striking laboratory findings were marked lymphopenia and thrombocytopenia. Elevated cytokine levels have been observed in patients and such excessive cytokine responses may contribute to the clinical severity of H7N9 infection. 9 Originating from reassortment events involving at least three avian influenza viruses, H7N9 viruses with multiple genotypes continue to emerge on a more frequent basis in 2014.", "9 Originating from reassortment events involving at least three avian influenza viruses, H7N9 viruses with multiple genotypes continue to emerge on a more frequent basis in 2014. Many viruses isolated from humans contain the E627K amino acid substitution in the polymerase PB2 component, associated with mammalian adaptation, and the G186V and Q226L substitutions in the haemagglutinin (HA) that are associated with dual receptor binding to both a2,3 and a2,6-linked sialic acid receptors. 10 All H7N9 viruses from the outbreak to date are antigenically similar to the (original) candidate vaccine strain.", "10 All H7N9 viruses from the outbreak to date are antigenically similar to the (original) candidate vaccine strain. Prototype inactivated whole particle H7N9 vaccines have been investigated in macaques and shown to induce good antibody responses that significantly reduced the number of days of virus shedding in experimentally infected animals. Treatment of H7N9-infected patients with neuraminidase inhibitors (NAIs), including intravenous (IV) peramivir or zanamivir, 11 appears to have been beneficial even when therapy was started late, although emergence of oseltamivir resistance has been associated with poor clinical outcomes.", "Treatment of H7N9-infected patients with neuraminidase inhibitors (NAIs), including intravenous (IV) peramivir or zanamivir, 11 appears to have been beneficial even when therapy was started late, although emergence of oseltamivir resistance has been associated with poor clinical outcomes. 12 All H7N9 viruses are amantadine-resistant due to the S31N substitution in the M2 ion channel protein, while viruses containing the R292K substitution in neuraminidase (NA), which confers resistance to both oseltamivir and peramivir (>1000-fold rise in IC 50 ) and reduced susceptibility to zanamivir and laninamivir (50-and 25-fold rises in IC 50 , respectively), have been reported in six cases. Two of these patients with severe H7N9 infection requiring extracorporeal membrane oxygenation (ECMO) also received systemic corticosteroid treatment leading to treatment failure and a poor clinical outcome.", "Two of these patients with severe H7N9 infection requiring extracorporeal membrane oxygenation (ECMO) also received systemic corticosteroid treatment leading to treatment failure and a poor clinical outcome. 12 The replication and transmission of H7N9 viruses containing the R292K NA mutation have been shown to be comparable to those of wild-type H7N9 viruses in guinea pigs, 13 and in ferrets following both contact and non-contact exposure. However, the wild-type virus did outgrow the R292K-resistant strain in some ferrets over the course of the infection.", "However, the wild-type virus did outgrow the R292K-resistant strain in some ferrets over the course of the infection. 14 Interestingly, the R292K variant appeared to be the dominant virus in ferret lung lobes, while in nasal turbinates, the wild-type virus was predominant. Therefore, it appears that the R292K mutation causes less fitness loss in H7N9 virus than in seasonal H3N2 viruses.", "Therefore, it appears that the R292K mutation causes less fitness loss in H7N9 virus than in seasonal H3N2 viruses. 13\n\nA new reassortant genotype of H5N1 containing the HA and NA genes from clade 1.1.2 and the internal genes from clade 2.3.2.1 emerged during 2013 and was associated with the highest number of cases (n = 26) and deaths (n = 14) in Cambodia. 15 Globally since 2003, there have been 650 confirmed H5N1 cases and 386 deaths reported in humans, 16 with most infections in the last 2 years being in children.", "15 Globally since 2003, there have been 650 confirmed H5N1 cases and 386 deaths reported in humans, 16 with most infections in the last 2 years being in children. Human-to-human transmission remains extremely rare based on virological and serological data from analysis of close contacts of confirmed cases in Cambodia, Thailand and Vietnam. 17 A study of household transmission patterns in Indonesia has shown that the overall household attack rate was 18Á3% and the secondary attack rate was 5Á5%, independent of household size.", "17 A study of household transmission patterns in Indonesia has shown that the overall household attack rate was 18Á3% and the secondary attack rate was 5Á5%, independent of household size. 18 Oseltamivir therapy appears to reduce mortality when administered within 8 days of H5N1 illness onset, although earlier treatment is more effective, highlighting the need for early patient diagnosis. 19 Early initiation of oseltamivir was particularly effective in reducing mortality in H5N1 patients without respiratory failure (odds ratio, 0Á17; P = 0Á04), whereas those requiring ventilatory support at the time of oseltamivir initiation were more likely to die.", "19 Early initiation of oseltamivir was particularly effective in reducing mortality in H5N1 patients without respiratory failure (odds ratio, 0Á17; P = 0Á04), whereas those requiring ventilatory support at the time of oseltamivir initiation were more likely to die. 20 A study of the risk factors for mortality related to H5N1 identified age, country, per capita government health expenditure and delay from symptom onset to hospitalisation as the key parameters, highlighting the importance of early diagnosis, treatment and supportive care. 21 High-dose systemic corticosteroids (SC) are associated with worse outcomes in H5N1 patients.", "21 High-dose systemic corticosteroids (SC) are associated with worse outcomes in H5N1 patients. 22 One human case of avian H5N6 was recently detected in China; the virus was a reassortant that contained seven genes from H5N1 and the NA gene from an H6N6 virus circulating in ducks. 23 China has also reported the detection of three human infections, two fatal, with avian H10N8 viruses that contain the internal genes from H9N2, as does H7N9.", "23 China has also reported the detection of three human infections, two fatal, with avian H10N8 viruses that contain the internal genes from H9N2, as does H7N9. 24 Like the H7N9 virus, the H10N8 virus has low pathogenicity in poultry and is therefore difficult to detect in birds. Burden in target populations Pregnant women and infants have an increased risk of complications following influenza infection.", "Burden in target populations Pregnant women and infants have an increased risk of complications following influenza infection. Globally, significant numbers of pregnant women died during the 2009-2010 pandemic, but no maternal mortality occurred in Japan. 25 Through education campaigns directed at pregnant women and healthcare professionals, 67% of pregnant women were vaccinated against H1N1pdm09 resulting in an infection rate among pregnant women in Japan of 3Á5% compared to the overall infection rate in the population of 12%.", "25 Through education campaigns directed at pregnant women and healthcare professionals, 67% of pregnant women were vaccinated against H1N1pdm09 resulting in an infection rate among pregnant women in Japan of 3Á5% compared to the overall infection rate in the population of 12%. 26 Of those pregnant women who were infected with H1N1pdm09 in Japan, 95% were treated with antivirals, and importantly, 88% of those were treated within 2 days of symptom onset. 25 In Mongolia, a prospective cohort study during 2013-2014 found that influenza-like illness (ILI) was detected in 17Á9% of pregnant women, of whom the majority tested positive for influenza A, with substantially lower influenza B and respiratory syncytial virus (RSV) infection.", "25 In Mongolia, a prospective cohort study during 2013-2014 found that influenza-like illness (ILI) was detected in 17Á9% of pregnant women, of whom the majority tested positive for influenza A, with substantially lower influenza B and respiratory syncytial virus (RSV) infection. 27 During the same period, ILI was detected in 30Á9% of infants <6 months of age, with an even spread of influenza A, influenza B and RSV. 27 The influenza burden in children in a rural Indian community was found to be substantial with 11Á6% of ILI cases being caused by influenza A or B viruses.", "27 The influenza burden in children in a rural Indian community was found to be substantial with 11Á6% of ILI cases being caused by influenza A or B viruses. 28 These findings underscore the importance of maternal immunisation. 29 Transmission patterns Sequence analysis of influenza viruses isolated from students on a Singapore University Campus provided insights into the chain of transmission, showing that 62% of 32 viruses were highly similar, demonstrating that the majority of transmission was occurring on the university campus rather than from infections outside.", "29 Transmission patterns Sequence analysis of influenza viruses isolated from students on a Singapore University Campus provided insights into the chain of transmission, showing that 62% of 32 viruses were highly similar, demonstrating that the majority of transmission was occurring on the university campus rather than from infections outside. 30 The effectiveness of surgical masks, hand hygiene and health education investigated in households in Hong Kong and Bangkok detected no significant difference in attack rate in cohorts using one of these interventions. 31 Further analysis of the data enabled some insights into the relative importance of aerosol, large droplet and contact transmission within the households.", "31 Further analysis of the data enabled some insights into the relative importance of aerosol, large droplet and contact transmission within the households. For influenza A infections, aerosol transmission appeared to be the most common route, whereas contact transmission caused the highest number of influenza B infections. 31 \n\nUse and effectiveness Neuraminidase inhibitors are commonly used for the treatment of influenza in Japan, typically following a positive result from a point-of-care (POC) test.", "31 \n\nUse and effectiveness Neuraminidase inhibitors are commonly used for the treatment of influenza in Japan, typically following a positive result from a point-of-care (POC) test. During the 2009-2010 pandemic, over 20 million POC test kits were shipped to hospitals and clinics in Japan to enable rapid diagnosis, and 89% of treated cases were administered NAIs within 48 hours of symptom onset. 32 In Japan during 2013, oseltamivir and laninamivir each represented 40% of NAIs used, while zanamivir (15%) and peramivir (5%) use was considerably less.", "32 In Japan during 2013, oseltamivir and laninamivir each represented 40% of NAIs used, while zanamivir (15%) and peramivir (5%) use was considerably less. NAI effectiveness has been assessed in numerous observational studies in Japan. Oseltamivir effectiveness is significantly reduced in patients with delayed treatment, and duration of fever and viral shedding is longer in treated patients with influenza B compared to influenza A virus infections.", "Oseltamivir effectiveness is significantly reduced in patients with delayed treatment, and duration of fever and viral shedding is longer in treated patients with influenza B compared to influenza A virus infections. 33, 34 The reduced effectiveness against influenza B viruses was also observed in zanamivir 33, 35 and laninamivir 35 trials. To determine whether NAIs reduced mortality during the 2009-2010 pandemic, data were compiled on 29 234 patients hospitalised with confirmed A(H1N1)pdm09 infection.", "To determine whether NAIs reduced mortality during the 2009-2010 pandemic, data were compiled on 29 234 patients hospitalised with confirmed A(H1N1)pdm09 infection. 36 Compared with no treatment, NAI treatment was associated with significantly reduced mortality, with early treatment also showing a reduced risk of mortality compared to late treatment. Although there was no significant clinical effect when comparing late treatment with no treatment in hospitalised patients, there was a significant benefit in treating patients who arrive late into intensive care units.", "Although there was no significant clinical effect when comparing late treatment with no treatment in hospitalised patients, there was a significant benefit in treating patients who arrive late into intensive care units. 36 In a household prophylaxis study, inhaled laninamivir given for either 2 or 3 days reduced the illness rate within households to 3Á9% and 3Á7%, respectively, compared to 16Á9% in households given a placebo. 37 A ferret model of oseltamivir prophylaxis has shown that while morbidity was significantly reduced, the prophylaxis regimes did not prevent infection nor significantly reduce virus load.", "37 A ferret model of oseltamivir prophylaxis has shown that while morbidity was significantly reduced, the prophylaxis regimes did not prevent infection nor significantly reduce virus load. 38 Resistance Although all four NAIs are sialic acid analogues, they have subtle differences in chemical structure and binding properties. Consequently, resistance patterns vary across NAIs.", "Consequently, resistance patterns vary across NAIs. The most commonly detected NA substitution causing NAI resistance in N1-containing influenza viruses is H275Y, which confers resistance to oseltamivir and peramivir, but not to zanamivir and laninamivir. This resistance mutation became fixed in seasonal H1N1 viruses circulating in 2008-2009.", "This resistance mutation became fixed in seasonal H1N1 viruses circulating in 2008-2009. 39 A late 2013 cluster of H1N1pdm09 viruses containing the H275Y substitution was detected in 38 (39%) of 97 H1N1pdm09 viruses from community patients not receiving NAIs in Sapporo, Japan, 40 reminiscent of a similar cluster of oseltamivir-resistant H1N1pdm09 viruses in community patients in Australia in 2011. 41, 42 Importantly, both sets of viruses contained permissive NA mutations (V241I and N369K) that have been shown in ferret studies to offset the destabilising and negative effect of the H275Y NA mutation.", "41, 42 Importantly, both sets of viruses contained permissive NA mutations (V241I and N369K) that have been shown in ferret studies to offset the destabilising and negative effect of the H275Y NA mutation. 43 In hospitalised influenza patients being treated with intravenous zanamivir, next-generation sequencing has been utilised to identify minor resistant virus populations. A total of five NA substitutions were identified in different viruses, including E119K and E119D; however, all apart from E119D were present in such low proportions that they could not be detected by Sanger 'population' sequencing methods.", "A total of five NA substitutions were identified in different viruses, including E119K and E119D; however, all apart from E119D were present in such low proportions that they could not be detected by Sanger 'population' sequencing methods. 44 The effects of various mutations in catalytic and framework residues of influenza B NA were investigated using reverse genetics and a range of functional assays. Four substitutions (D198E, I222T, H274Y and N294S) conferred reduced susceptibility to oseltamivir, while three substitutions (E119A, D198Y and R371K) caused highly reduced inhibition by oseltamivir, zanamivir and peramivir.", "Four substitutions (D198E, I222T, H274Y and N294S) conferred reduced susceptibility to oseltamivir, while three substitutions (E119A, D198Y and R371K) caused highly reduced inhibition by oseltamivir, zanamivir and peramivir. 45 Two of these variants (H274Y, E119A) had in vitro replication fitness comparable to the NAI-susceptible viruses. To date, these substitutions have only been detected on rare occasions in circulating influenza viruses.", "To date, these substitutions have only been detected on rare occasions in circulating influenza viruses. An intravenous formulation of zanamivir showed both virological and clinical effectiveness without safety concerns in patients hospitalised with influenza in Japan. 46 A range of new adamantane derivatives have good antiviral activity in vitro and in animal models against H1N1pdm09 and H3N2 viruses that contain the S31N M2 ion channel substitution that confers resistance to amantadine.", "46 A range of new adamantane derivatives have good antiviral activity in vitro and in animal models against H1N1pdm09 and H3N2 viruses that contain the S31N M2 ion channel substitution that confers resistance to amantadine. 47 Favipiravir is a novel pyrazinamide molecule that inhibits replication of various RNA viruses, including influenza types A, B and C (including oseltamivir-resistant strains), and has recently been licensed in Japan for the control of novel or reemerging influenza viruses. Its triphosphate metabolite is an RNA polymerase inhibitor which disrupts virus genome replication; synergy with oseltamivir has been demonstrated in pre-clinical models.", "Its triphosphate metabolite is an RNA polymerase inhibitor which disrupts virus genome replication; synergy with oseltamivir has been demonstrated in pre-clinical models. 48 A phase II study in the US has shown that a twice-daily regimen decreased the titre and time to cessation of virus shedding, and had a significant benefit in reducing clinical symptoms (NCT01068912; .gov). Subsequent phase III studies are currently ongoing (NCT02008344 and NCT02026349).", "Subsequent phase III studies are currently ongoing (NCT02008344 and NCT02026349). A neutralising monoclonal antibody (MHAA4549A) which binds to the HA stalk of influenza A viruses in both group 1 and group 2 HA subtypes has been effective when given up to 72 hours post-infection in mice and ferrets infected with H5N1. 49 Phase I and IIa trials in humans showed that the antibody was well tolerated, had a mean half-life of 21Á9 days and was effective as therapy at high doses in experimentally infected volunteers (NCT01877785).", "49 Phase I and IIa trials in humans showed that the antibody was well tolerated, had a mean half-life of 21Á9 days and was effective as therapy at high doses in experimentally infected volunteers (NCT01877785). Upcoming placebo-controlled phase IIb trials will target hospitalised influenza patients requiring oxygen and compare the combination of the monoclonal antibody with oseltamivir to oseltamivir monotherapy (NCT01980966). Other broadly neutralising antibodies against multiple clades of H5N1 have been generated by glycan masking of key HA antigenic residues to direct antibody responses to the more conserved stem region of the HA.", "Other broadly neutralising antibodies against multiple clades of H5N1 have been generated by glycan masking of key HA antigenic residues to direct antibody responses to the more conserved stem region of the HA. 50 FluPep, a novel peptide that prevents virus entry into cells, has been shown in mouse studies to be effective in reducing virus titres in lungs, inflammatory cytokines and mortality. 51 Fludase (DAS-181) is a host-targeted therapeutic agent that removes sialic acid from cellular receptors in the respiratory tract, thus preventing influenza virus binding.", "51 Fludase (DAS-181) is a host-targeted therapeutic agent that removes sialic acid from cellular receptors in the respiratory tract, thus preventing influenza virus binding. Delivered topically, it is effective in animal models of lethal H5N1 and H7N9 infection, including a NAI-resistant R292K H7N9 variant. 52 In a phase 2 RCT, inhaled DAS181 reduced pharyngeal viral replication in uncomplicated influenza but did not reduce nasal virus loads or improve clinical outcomes.", "52 In a phase 2 RCT, inhaled DAS181 reduced pharyngeal viral replication in uncomplicated influenza but did not reduce nasal virus loads or improve clinical outcomes. 53 Another receptor-targeted approach is the development of multivalent sialic acid-binding proteins; 54 a single administration 7 days pre-infection resulted in the protection of 80-100% of mice from lethal H7N9 challenge. 55 Apart from blocking sialic acid, the compound appears to stimulate the expression of pro-inflammatory mediators, thereby 'preparing' the immune system for subsequent influenza infection.", "55 Apart from blocking sialic acid, the compound appears to stimulate the expression of pro-inflammatory mediators, thereby 'preparing' the immune system for subsequent influenza infection. When delivered 24 hours post-infection, protection was, however, only 20-40%. 55 Although drug resistance is considered less likely to occur with host-directed therapies, escape mutants have developed rapidly following exposure to a host-directed vacuolar ATPase-inhibiting drug.", "55 Although drug resistance is considered less likely to occur with host-directed therapies, escape mutants have developed rapidly following exposure to a host-directed vacuolar ATPase-inhibiting drug. 56 Furthermore, following serial passage of different viruses in the presence of bafilomycin A1, two HA mutations were selected (A19T and S210N) which resulted in reduced drug susceptibility and increased virulence in mice. 56 \n\nMultiple influenza vaccine effectiveness (IVE) studies have used the control test negative design approach to estimate IVE during early and late phases of influenza seasons, the 2009 pandemic, and by age or target groups.", "56 \n\nMultiple influenza vaccine effectiveness (IVE) studies have used the control test negative design approach to estimate IVE during early and late phases of influenza seasons, the 2009 pandemic, and by age or target groups. Typically, IVE estimates range from 40% to 60% each season. 57 Future studies will investigate IVE with respect to the type of influenza vaccine used, whether IVE differs between the start and end of the season and the effect of previous vaccination.", "57 Future studies will investigate IVE with respect to the type of influenza vaccine used, whether IVE differs between the start and end of the season and the effect of previous vaccination. In Japan in 2013/14, IVE for influenza A in children aged 1-5 years averaged 72% (95% CI 64-79), dropped to 48% (95% CI 31-61) in children aged 6-12 years and was not apparent against influenza B in any age group (À1%, 95% CI À19 to 14). 58 Influenza vaccine effectiveness is known to be lower in adults over 65 years of age, a group that accounts for >60% of seasonal influenza-related hospitalisations and >90% of influenza-related deaths.", "58 Influenza vaccine effectiveness is known to be lower in adults over 65 years of age, a group that accounts for >60% of seasonal influenza-related hospitalisations and >90% of influenza-related deaths. In an effort to improve IVE in the elderly, recent RCTs have investigated the use of adjuvants, intradermal injection and higher doses of antigen. While the use of AS03-adjuvanted influenza vaccine was only moderately superior to non-adjuvanted vaccine in the elderly, 59 the use of a high-dose vaccine containing four times the standard level of HA (60 lg per virus) did result in improved effectiveness compared to the standard dose vaccine.", "While the use of AS03-adjuvanted influenza vaccine was only moderately superior to non-adjuvanted vaccine in the elderly, 59 the use of a high-dose vaccine containing four times the standard level of HA (60 lg per virus) did result in improved effectiveness compared to the standard dose vaccine. 60 For vaccine manufacturers, generating high-growth reassortants of certain circulating viruses can be challenging. A recent study used random mutagenesis of PR8 and selection of high-growth clones in MDCK and Vero cells to derive a high-growth version of PR8.", "A recent study used random mutagenesis of PR8 and selection of high-growth clones in MDCK and Vero cells to derive a high-growth version of PR8. 61 Reassortment of the highgrowth PR8 virus with the HA/NA of either H5N1, H7N9 or seasonal influenza viruses showed that yields significantly exceeded equivalent reassortants that contained the internal genes of the 'normal' PR8 virus. 61 \n\nAs of July 2014, the number of confirmed cases of MERS-CoV has exceeded 830, with at least 288 associated deaths.", "61 \n\nAs of July 2014, the number of confirmed cases of MERS-CoV has exceeded 830, with at least 288 associated deaths. 62 The majority of cases have involved patients with comorbidities (76%) and are predominately males (63%) with a median age of 47. 63, 64 Fewer than 25% of patients have reported contact with animals including dromedary camels, which have been shown to be one likely animal reservoir based on sero-positivity and detection of MERS-CoV.", "63, 64 Fewer than 25% of patients have reported contact with animals including dromedary camels, which have been shown to be one likely animal reservoir based on sero-positivity and detection of MERS-CoV. 65 More than 25% of the infections have been in healthcare workers, and the large number of nosocomial infections is likely due to inadequate infection control in hospitals plus enhanced surveillance that has detected a substantial number of mild or asymptomatic infections. 63 Outside hospital, the burden of disease is likely to be larger than has been reported.", "63 Outside hospital, the burden of disease is likely to be larger than has been reported. 66 Serological analysis of several UK patients found a rapid rise in antibodies from day 10, and that titres were maintained for at least 300 days post-infection. Anti-S (spike glycoprotein) antibodies are responsible for virus neutralisation.", "Anti-S (spike glycoprotein) antibodies are responsible for virus neutralisation. Importantly for serological analyses, patients who experience only mild disease may mount only a modest serological response. 67 Sequential samples from three cases involved in a chain of transmission were extensively analysed using next-generation sequencing.", "67 Sequential samples from three cases involved in a chain of transmission were extensively analysed using next-generation sequencing. 68 Various minority variants were detected, of which some were transient while others were transmitted, and there was evidence of variation in frequency of some variants in different body compartments. Various therapeutic options have been investigated for the treatment of MERS-CoV, but no therapy of proven value currently exists.", "Various therapeutic options have been investigated for the treatment of MERS-CoV, but no therapy of proven value currently exists. The use of SC was associated with adverse outcome in SARS 69 and is not recommended for MERS-CoV. Many agents have shown inhibitory effects against MERS-CoV in cell culture including interferon +/À ribavirin, cyclosporine A, mycophenolic acid, chloroquine and lopinavir.", "Many agents have shown inhibitory effects against MERS-CoV in cell culture including interferon +/À ribavirin, cyclosporine A, mycophenolic acid, chloroquine and lopinavir. 70 Interferons, lopinavir, mycophenolate, possibly alisporivir and combinations are reasonable choices for testing in controlled clinical trials. Exploratory post hoc metaanalysis of studies related to SARS and severe influenza has shown a significant reduction in mortality following convalescent plasma treatment compared to placebo or no therapy (odds ratio 0Á25; 95% CI 0Á14-0Á45).", "Exploratory post hoc metaanalysis of studies related to SARS and severe influenza has shown a significant reduction in mortality following convalescent plasma treatment compared to placebo or no therapy (odds ratio 0Á25; 95% CI 0Á14-0Á45). 71 Thus, the early use of virus-specific neutralising antibodies in the form of convalescent plasma and monoclonal or polyclonal neutralising antibodies for treatment of MERS-CoV has the highest likelihood of clinical benefit. 64 Modalities with risks likely to exceed benefits include SC, ribavirin monotherapy and IVIG.", "64 Modalities with risks likely to exceed benefits include SC, ribavirin monotherapy and IVIG. 72\n\nRespiratory syncytial virus (RSV) Respiratory syncytial virus disproportionately impacts children in low-income countries. 73 Almost all children will have been infected with RSV by their 2nd birthday, and it is the number one cause of hospitalisation of infants in the US, causing 10 times more infant deaths than influenza.", "73 Almost all children will have been infected with RSV by their 2nd birthday, and it is the number one cause of hospitalisation of infants in the US, causing 10 times more infant deaths than influenza. 74 In addition, RSV infects 3-10% of adults annually and accounts for 5-15% of community acquired pneumonia (CAP) and 9-10% of hospitalisations, 75 a burden of disease that approaches that caused by influenza. In a study, conducted in Hong Kong, of 607 hospitalised adults with RSV, 40% had pneumonia and 70% required supplementary oxygen; mortality rates and duration of hospital stay were similar to those observed for influenza patients.", "In a study, conducted in Hong Kong, of 607 hospitalised adults with RSV, 40% had pneumonia and 70% required supplementary oxygen; mortality rates and duration of hospital stay were similar to those observed for influenza patients. 75 Approximately, 15% of hospitalised RSV patients had bacterial superinfections. Although corticosteroids were used to treat 38% of patients, treatment had no benefit on clinical outcome, and instead increased bacterial secondary infections and caused a longer duration of illness.", "Although corticosteroids were used to treat 38% of patients, treatment had no benefit on clinical outcome, and instead increased bacterial secondary infections and caused a longer duration of illness. 75 RSV replication appears prolonged in patients with comorbidities and LRT complications. Palivizumab prophylaxis of premature infants of <6 months of age has been shown to reduce hospitalisation due to RSV by 55%.", "Palivizumab prophylaxis of premature infants of <6 months of age has been shown to reduce hospitalisation due to RSV by 55%. 76 Preventing RSV during infancy has been associated with reduction of wheezing later in life. 77 Trials of other monoclonal antibodies have typically shown that they do not achieve superiority compared to palivizumab and therefore do achieve licensure.", "77 Trials of other monoclonal antibodies have typically shown that they do not achieve superiority compared to palivizumab and therefore do achieve licensure. Furthermore, treatment with neutralising monoclonal antibodies does not appear to reduce virus load or disease severity in hospitalised infants. 78 Alternative options for RSV therapy to be assessed in future clinical trials include inhaled nanobodies, aerosolised peptides, nucleoside analogues and RNA-interference molecules.", "78 Alternative options for RSV therapy to be assessed in future clinical trials include inhaled nanobodies, aerosolised peptides, nucleoside analogues and RNA-interference molecules. Human rhinoviruses (HRV) usually cause mild acute respiratory infections, but on occasions can also cause more severe respiratory infections, including exacerbations of asthma and COPD. Of 115 Japanese children with asthma, a respiratory virus was detected in 86%, of which HRV (n = 36) or RSV (n = 47) were most common.", "Of 115 Japanese children with asthma, a respiratory virus was detected in 86%, of which HRV (n = 36) or RSV (n = 47) were most common. 79 Ex vivo bronchial epithelial cells from people with asthma are more susceptible to HRV infection, due to deficient induction of IFN-b and IFN-lambda. In a study of 147 asthmatics on inhaled corticosteroid therapy, with a history of virusassociated exacerbations, patients were randomised to 14-day treatment with inhaled IFN-b or placebo within 24 hours of developing cold symptoms.", "In a study of 147 asthmatics on inhaled corticosteroid therapy, with a history of virusassociated exacerbations, patients were randomised to 14-day treatment with inhaled IFN-b or placebo within 24 hours of developing cold symptoms. Patients who received IFN-b had enhanced morning peak expiratory flow recovery, reduced need for additional treatment and boosted innate immunity as assessed by blood and sputum biomarkers. In an exploratory analysis of a subset of more difficult-to-treat asthma (n = 27 IFN-b; n = 31 placebo), worsening of symptoms increased significantly in the placebo group, but was prevented by IFN-b (P = 0Á004).", "In an exploratory analysis of a subset of more difficult-to-treat asthma (n = 27 IFN-b; n = 31 placebo), worsening of symptoms increased significantly in the placebo group, but was prevented by IFN-b (P = 0Á004). 80 A picornavirus-specific antiviral, vapendavir, was found to reduce symptom scores, lower bronchodilator puffer use and reduce viral load in asthma patients with an URTI due to HRV. 81 \n\nDiagnostics Point-of-care tests that can deliver a result in 15 minutes have been available in many countries for the last decade, but while having good specificity, the sensitivity has typically been poor, ranging from 10% to 80% compared to PCR or culture.", "81 \n\nDiagnostics Point-of-care tests that can deliver a result in 15 minutes have been available in many countries for the last decade, but while having good specificity, the sensitivity has typically been poor, ranging from 10% to 80% compared to PCR or culture. Their use in emergency departments of hospitals can result in reduced unnecessary antibiotic use and an increased likelihood of discharge. Newer immunofluorescence-based POC tests with improved sensitivity are being developed.", "Newer immunofluorescence-based POC tests with improved sensitivity are being developed. In addition, the Quidel Sofia POC test may be linked via the internet such that results can be reported in real-time to central databases. Other POC tests are using photographic silver amplification immunochromatography technology to increase sensitivity.", "Other POC tests are using photographic silver amplification immunochromatography technology to increase sensitivity. 82 PCR remains the gold standard for virus diagnostics with an ability to be rapid, sensitive, specific and to identify a wide range of pathogens via different assays. The ability to multiplex multiple pathogen targets allows costs to be reduced in a diagnostic setting.", "The ability to multiplex multiple pathogen targets allows costs to be reduced in a diagnostic setting. New closed-system technologies which involve only minimal hands-on time (a few minutes) and that conduct both automated nucleic acid extraction and PCR for multiple pathogens are now available, but are currently limited for clinical diagnostic purposes due to low-throughput capabilities. 83 Next-generation sequencing technologies and PCR-based analyses with increasing sensitivity both offer considerable scope in diagnosis, although our current understanding of the clinical impact of pathogens at low levels or the presence of variants as minor virus populations is limited.", "83 Next-generation sequencing technologies and PCR-based analyses with increasing sensitivity both offer considerable scope in diagnosis, although our current understanding of the clinical impact of pathogens at low levels or the presence of variants as minor virus populations is limited. Providing low-cost, sensitive assays for diagnosing respiratory virus infections in low/middle-income countries is challenging, but has the potential to improve treatment and avoid unnecessary antibiotic use in these regions. Repurposed drugs for respiratory viral infections Nitazoxanide (NTX) is an antiparasitic agent approved for Giardia and Cryptosporidium infections that also inhibits replication in vitro of influenza and other respiratory viruses.", "Repurposed drugs for respiratory viral infections Nitazoxanide (NTX) is an antiparasitic agent approved for Giardia and Cryptosporidium infections that also inhibits replication in vitro of influenza and other respiratory viruses. 84 Treatment with NTX 600 mg twice daily for 5 days was associated with a reduction in the duration of symptoms in participants with acute uncomplicated influenza. 85 In a subset analysis of 238 patients with no confirmed virus infection, treatment with NTX 600 mg also led to a shorter time to alleviation of symptoms in comparison to placebo (88Á4 versus 105Á7 hours, P = 0Á02).", "85 In a subset analysis of 238 patients with no confirmed virus infection, treatment with NTX 600 mg also led to a shorter time to alleviation of symptoms in comparison to placebo (88Á4 versus 105Á7 hours, P = 0Á02). 86 Systemic corticosteroids for respiratory virus infections A review of prospective observational studies has shown that SC increased the risks of mortality and morbidity (e.g. secondary infections, hospital-acquired pneumonia) in severe infection due to influenza A(H1N1)pdm09 especially with delayed antiviral therapy.", "secondary infections, hospital-acquired pneumonia) in severe infection due to influenza A(H1N1)pdm09 especially with delayed antiviral therapy. 87 During SARS infections, a higher risk of avascular necrosis and prolonged virus shedding were observed in patients who had received highdose SC therapy. 88 It is therefore important to avoid the use of high-dose SC in severe respiratory viral infections outside the context of clinical trials.", "88 It is therefore important to avoid the use of high-dose SC in severe respiratory viral infections outside the context of clinical trials. Larger trials are needed to resolve the uncertainty regarding the effect of early SC therapy in ARDS. Low-dose SC is indicated for management of refractory septic shock, 89 and a short course of SC is indicated for acute exacerbations of obstructive airway diseases (asthma, COPD) 90 Current evidence does not support a clinically relevant effect of systemic or inhaled glucocorticoids on admission or length of hospitalisation for acute viral bronchiolitis in infants and young children.", "Low-dose SC is indicated for management of refractory septic shock, 89 and a short course of SC is indicated for acute exacerbations of obstructive airway diseases (asthma, COPD) 90 Current evidence does not support a clinically relevant effect of systemic or inhaled glucocorticoids on admission or length of hospitalisation for acute viral bronchiolitis in infants and young children. 91 \n\nRespiratory syncytial virus, influenza viruses, parainfluenza (PIV) viruses and adenoviruses (AdVs) cause the most serious disease in immunocompromised hosts, but other respiratory viruses are becoming increasingly appreciated as a cause of both upper and lower respiratory tract disease. The potential for these viruses to cause lower respiratory tract infections (LRTI) after transplantation varies.", "The potential for these viruses to cause lower respiratory tract infections (LRTI) after transplantation varies. Human metapneumovirus infections have similar outcomes to RSV infection in hematopoietic stem cell transplant (HSCT) recipients, including potentially severe and fatal pneumonia. HRV and coronavirus infections are very frequent in transplant recipients, but severe lower respiratory tract disease is uncommon.", "HRV and coronavirus infections are very frequent in transplant recipients, but severe lower respiratory tract disease is uncommon. In a prospective study of 112 lung transplant recipients, the virus infection rates upon screening, routine and emergency visits were 14%, 15% and 34%, respectively. Picornaviruses were identified most frequently in nasopharyngeal (85/140; 61%) and BAL specimens (20/34; 59%).", "Picornaviruses were identified most frequently in nasopharyngeal (85/140; 61%) and BAL specimens (20/34; 59%). Asymptomatic virus carriage, mainly of picornaviruses, was found at 10% of screening visits. Infections were associated with transient lung function loss and high calcineurin inhibitor blood levels.", "Infections were associated with transient lung function loss and high calcineurin inhibitor blood levels. The hospitalisation rate was 50% for influenza and PIV and 16Á9% for other viruses. Acute rejection was not associated with virus infection.", "Acute rejection was not associated with virus infection. 92 The risk factors for severe LRTI among transplant recipients include early onset post-transplant (<3 m), steroid boluses, young children (<1 year), chronic GVHD, lymphopenia/lymphodepletion and allogeneic HSCT patients. 92 Influenza Immunocompromised patients with influenza exhibit more complications, longer virus shedding and more antiviral resistance, while often demonstrating milder clinical symptoms and signs on initial clinical assessment.", "92 Influenza Immunocompromised patients with influenza exhibit more complications, longer virus shedding and more antiviral resistance, while often demonstrating milder clinical symptoms and signs on initial clinical assessment. 93 Influenza A (H1N1)pdm09 viruses have the potential for rapid emergence of oseltamivir resistance and causing severe morbidity, particularly in immunocompromised patients with lymphopenia and delayed antiviral therapy. 94 Influenza viraemia may serve as a marker for overall poor outcome with increased risk of progression to LRTI, hypoxaemia, respiratory failure and death.", "94 Influenza viraemia may serve as a marker for overall poor outcome with increased risk of progression to LRTI, hypoxaemia, respiratory failure and death. Influenza RNA in blood (viraemia) was detected in nine of 79 (11Á4%) HSCT recipients with influenza. Among patients with LRTI, viraemia was associated with increased hazards of overall as well as influenzaassociated death (hazard ratio 3Á5, 1Á1-12).", "Among patients with LRTI, viraemia was associated with increased hazards of overall as well as influenzaassociated death (hazard ratio 3Á5, 1Á1-12). 95 In 143 HSCT recipients with documented seasonal influenza infection, treatment with high-dose corticosteroids was associated with a trend towards prolonged virus shedding [(OR), 3Á3; 95% CI 1Á0-11; P = 0Á05], whereas antiviral therapy initiated to treat upper respiratory tract infection (URTI) was associated with fewer cases of LRTI (OR, 0Á04; 95% CI, 0-0Á2; P < 0Á01) and fewer hypoxaemia episodes (OR, 0Á3; 95% CI, 0Á1-0Á9; P = 0Á03). 96 In view of the risks of prolonged replication and drug resistance emergence, 97 a longer duration and a higher NAI dose may be beneficial.", "96 In view of the risks of prolonged replication and drug resistance emergence, 97 a longer duration and a higher NAI dose may be beneficial. Early therapy was consistently demonstrated to have improved outcomes. [98] [99] [100] [101] Other treatment options under study are the use of triple combination therapy with amantadine, oseltamivir and ribavirin, 102 or intravenous peramivir 103 or zanamivir.", "[98] [99] [100] [101] Other treatment options under study are the use of triple combination therapy with amantadine, oseltamivir and ribavirin, 102 or intravenous peramivir 103 or zanamivir. 104 Therapy of influenza in lung transplant recipients is associated with a reduced risk of developing bronchiolitis obliterans syndrome. 105 Parainfluenza DAS181 is inhibitory for PIV and influenza viruses, including those resistant to the amantadine and NAIs 106 and may be effective in treating immunocompromised patients with severe PIV lung disease.", "105 Parainfluenza DAS181 is inhibitory for PIV and influenza viruses, including those resistant to the amantadine and NAIs 106 and may be effective in treating immunocompromised patients with severe PIV lung disease. 107 In a study of four severely immunocompromised children with PIV disease treatment with DAS181 for 5-10 days, by dry powder inhalation or nebulisation, was well tolerated. Transient increase in serum alkaline phosphatase, liver function and coagulation tests were observed, but nasal wash virus loads were reduced in all patients within 1 week with improved clinical features.", "Transient increase in serum alkaline phosphatase, liver function and coagulation tests were observed, but nasal wash virus loads were reduced in all patients within 1 week with improved clinical features. 108 \n\nIn a RCT of lung transplant recipients with RSV infection, the incidence of new or progressive bronchiolitis obliterans syndrome at day 90 was significantly reduced in 16 patients who received a small interfering RNA against the RSV Ngene (ALN-RSV01) compared with placebo (n = 8) (6Á3% versus 50%, P = 0Á027). 109 In a larger follow-up multicentre phase IIb study, treatment with ALN-RSV01 showed a greater than eightfold reduced risk in developing bronchiolitis obliterans syndrome at day 180.", "109 In a larger follow-up multicentre phase IIb study, treatment with ALN-RSV01 showed a greater than eightfold reduced risk in developing bronchiolitis obliterans syndrome at day 180. 110\n\nAdenovirus is a serious, often fatal infection in immunocompromised patients, especially in HSCT recipients. The control of AdV is mostly T-cell mediated, and therefore, patients who have received T-cell suppressive regimens are at an increased risk for AdV infection.", "The control of AdV is mostly T-cell mediated, and therefore, patients who have received T-cell suppressive regimens are at an increased risk for AdV infection. The annual incidence of AdV infections in HSCT recipients ranges from 5% to 50%, and is increasing, likely due to increased use of T-celldepleted allografts and cord blood as source. The mortality rate is up to 80%.", "The mortality rate is up to 80%. 111 Brincidofovir (BCV; formerly CMX-001) is an orally bioavailable lipid-conjugate of cidofovir (CDV) that provides high intracellular concentrations of CDV diphosphate with a long intracellular half-life (up to 4-6Á5 days). BCV is 65-fold more potent against AdV than CDV in vitro with a low risk of myeloidor nephrotoxicity, but gastrointestinal side effects are more common.", "BCV is 65-fold more potent against AdV than CDV in vitro with a low risk of myeloidor nephrotoxicity, but gastrointestinal side effects are more common. 112 In a retrospective study of 13 immunocompromised patients given BCV for AdV disease after failing or intolerance to i.v. cidofovire nine patients (69Á2%) demonstrated a virological response (VR), which was defined as a 99% drop from baseline or undetectable AdV DNA in serum by week 8.", "cidofovire nine patients (69Á2%) demonstrated a virological response (VR), which was defined as a 99% drop from baseline or undetectable AdV DNA in serum by week 8. Patients with VR had longer survival than those without VR (median 196 days versus 54Á5 days; P = 0Á04). 113" ]

[ "This review highlights the main points which emerged from the presentations and discussions at the 3rd isirv-Antiviral Group Conference - advances in clinical management. The conference covered emerging and potentially pandemic influenza viruses and discussed novel/pre-licensure therapeutics and currently approved antivirals and vaccines for the control of influenza. Current data on approved and novel treatments for non-influenza respiratory viruses such as MERS-CoV, respiratory syncytial virus (RSV) and rhinoviruses and the challenges of treating immunocompromised patients with respiratory infections was highlighted.", "Current data on approved and novel treatments for non-influenza respiratory viruses such as MERS-CoV, respiratory syncytial virus (RSV) and rhinoviruses and the challenges of treating immunocompromised patients with respiratory infections was highlighted. Text: Recurrent infections by influenza and other respiratory viruses contribute enormously to the burden of human disease and (emergent) sporadic zoonotic infections, such as by influenza H7N9 and H5N1 and Middle East respiratory syndrome (MERS) coronavirus, pose a constant threat of a new global epidemic. Despite extensive knowledge of the viruses and their interaction with the host, there is little in our armoury of vaccines and therapeutics to combat this perpetual onslaught.", "Despite extensive knowledge of the viruses and their interaction with the host, there is little in our armoury of vaccines and therapeutics to combat this perpetual onslaught. The 3rd isirv Antiviral Group conference on Influenza and Other Respiratory Virus Infections: Advances in Clinical Management, convened in Tokyo, Japan on 4-6 June 2014, attracted 188 clinicians, public health specialists and medical scientists from 34 countries to present their recent research and discuss various aspects of the impact of respiratory viruses in different patient groups/settings and in different regions of the world. The programme 1 focused on the latest advances in the mitigation and clinical management of influenza and other respiratory virus disease, and the successful use of antivirals (and vaccines) against seasonal and pandemic influenza, particularly in Japan, as well as the development/assessment of novel antiviral agents.", "The programme 1 focused on the latest advances in the mitigation and clinical management of influenza and other respiratory virus disease, and the successful use of antivirals (and vaccines) against seasonal and pandemic influenza, particularly in Japan, as well as the development/assessment of novel antiviral agents. This overview highlights some of the main points which emerged from the presentations (both oral and poster) and associated discussion. In recent years, an increasing number of cases of novel animal influenza A viruses infecting humans have been reported.", "In recent years, an increasing number of cases of novel animal influenza A viruses infecting humans have been reported. These include multiple avian influenza A virus subtypes, in particular H5N1 and H7N9, and swine-origin H3N2v. Reasons for this increase include both social factors, for example, increased human populations living in close proximity to animals and increased surveillance and diagnostic testing.", "Reasons for this increase include both social factors, for example, increased human populations living in close proximity to animals and increased surveillance and diagnostic testing. Risk assessment tools have been developed by many authorities around the world (e.g. the European CDC, US CDC, USAID and WHO) to assist in predicting the likelihood that a particular virus will emerge and its associated impact, as well as prioritising the development of candidate human vaccine viruses.", "the European CDC, US CDC, USAID and WHO) to assist in predicting the likelihood that a particular virus will emerge and its associated impact, as well as prioritising the development of candidate human vaccine viruses. 2 These tools allow continual reassessment as new data become available and provide an objective, transparent process with which to make resource allocation and pandemic planning decisions. In February 2013, China detected the first human cases of H7N9 infection in severely ill patients with pneumonia.", "In February 2013, China detected the first human cases of H7N9 infection in severely ill patients with pneumonia. 3 As of May 22, 2014 , there have been 446 confirmed H7N9 cases in China resulting in 163 deaths. 4 The cases have occurred mainly during two waves (weeks [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 2013 and week 40, 2013week 20, 2014) , 4 of which 85% had prior exposure to poultry or contaminated live poultry markets.", "4 The cases have occurred mainly during two waves (weeks [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 2013 and week 40, 2013week 20, 2014) , 4 of which 85% had prior exposure to poultry or contaminated live poultry markets. The median time from poultry exposure to disease onset was 5 days, whereas the median time from illness onset to hospital admission, ARDS development, antiviral therapy and death was 5, 6Á8, 7 and 14 days, respectively. 5 Closure of live poultry markets has markedly reduced the risk of H7N9 infection.", "5 Closure of live poultry markets has markedly reduced the risk of H7N9 infection. Across nine areas in the two most affected provinces in China, modelling analysis estimated that the effectiveness of market closure was 97% (95% CI: 89%, 100%). 6 A retrospective serological study of blood specimens taken in January-May and October-November in 2012 from 1544 subjects who worked in live poultry markets, farms, slaughter houses or kept backyard poultry revealed no evidence of H7N9 infection, 7 indicating widespread population susceptibility and lack of prior circulation of antigenically related viruses.", "6 A retrospective serological study of blood specimens taken in January-May and October-November in 2012 from 1544 subjects who worked in live poultry markets, farms, slaughter houses or kept backyard poultry revealed no evidence of H7N9 infection, 7 indicating widespread population susceptibility and lack of prior circulation of antigenically related viruses. Multiple family clusters have been reported, but no sustained human-to-human transmission, with studies demonstrating a very low detection of virus or specific antibody in close contacts (0Á34% and 0Á2% in the first and second waves, respectively) and healthcare workers of positive cases. 8 In both the first and the second waves, the majority of the patients hospitalised with H7N9 infection were older men (median age, 62 and 58 years, respectively with an overall male/female ratio of 2Á2:1) and the case fatality was similar (32% and 39%, respectively).", "8 In both the first and the second waves, the majority of the patients hospitalised with H7N9 infection were older men (median age, 62 and 58 years, respectively with an overall male/female ratio of 2Á2:1) and the case fatality was similar (32% and 39%, respectively). Pre-existing medical conditions occurred in >60% of these cases. The prominent clinical features on admission were those of a severe influenza syndrome with fever, cough, fatigue and dyspnoea, while the most striking laboratory findings were marked lymphopenia and thrombocytopenia.", "The prominent clinical features on admission were those of a severe influenza syndrome with fever, cough, fatigue and dyspnoea, while the most striking laboratory findings were marked lymphopenia and thrombocytopenia. Elevated cytokine levels have been observed in patients and such excessive cytokine responses may contribute to the clinical severity of H7N9 infection. 9 Originating from reassortment events involving at least three avian influenza viruses, H7N9 viruses with multiple genotypes continue to emerge on a more frequent basis in 2014.", "9 Originating from reassortment events involving at least three avian influenza viruses, H7N9 viruses with multiple genotypes continue to emerge on a more frequent basis in 2014. Many viruses isolated from humans contain the E627K amino acid substitution in the polymerase PB2 component, associated with mammalian adaptation, and the G186V and Q226L substitutions in the haemagglutinin (HA) that are associated with dual receptor binding to both a2,3 and a2,6-linked sialic acid receptors. 10 All H7N9 viruses from the outbreak to date are antigenically similar to the (original) candidate vaccine strain.", "10 All H7N9 viruses from the outbreak to date are antigenically similar to the (original) candidate vaccine strain. Prototype inactivated whole particle H7N9 vaccines have been investigated in macaques and shown to induce good antibody responses that significantly reduced the number of days of virus shedding in experimentally infected animals. Treatment of H7N9-infected patients with neuraminidase inhibitors (NAIs), including intravenous (IV) peramivir or zanamivir, 11 appears to have been beneficial even when therapy was started late, although emergence of oseltamivir resistance has been associated with poor clinical outcomes.", "Treatment of H7N9-infected patients with neuraminidase inhibitors (NAIs), including intravenous (IV) peramivir or zanamivir, 11 appears to have been beneficial even when therapy was started late, although emergence of oseltamivir resistance has been associated with poor clinical outcomes. 12 All H7N9 viruses are amantadine-resistant due to the S31N substitution in the M2 ion channel protein, while viruses containing the R292K substitution in neuraminidase (NA), which confers resistance to both oseltamivir and peramivir (>1000-fold rise in IC 50 ) and reduced susceptibility to zanamivir and laninamivir (50-and 25-fold rises in IC 50 , respectively), have been reported in six cases. Two of these patients with severe H7N9 infection requiring extracorporeal membrane oxygenation (ECMO) also received systemic corticosteroid treatment leading to treatment failure and a poor clinical outcome.", "Two of these patients with severe H7N9 infection requiring extracorporeal membrane oxygenation (ECMO) also received systemic corticosteroid treatment leading to treatment failure and a poor clinical outcome. 12 The replication and transmission of H7N9 viruses containing the R292K NA mutation have been shown to be comparable to those of wild-type H7N9 viruses in guinea pigs, 13 and in ferrets following both contact and non-contact exposure. However, the wild-type virus did outgrow the R292K-resistant strain in some ferrets over the course of the infection.", "However, the wild-type virus did outgrow the R292K-resistant strain in some ferrets over the course of the infection. 14 Interestingly, the R292K variant appeared to be the dominant virus in ferret lung lobes, while in nasal turbinates, the wild-type virus was predominant. Therefore, it appears that the R292K mutation causes less fitness loss in H7N9 virus than in seasonal H3N2 viruses.", "Therefore, it appears that the R292K mutation causes less fitness loss in H7N9 virus than in seasonal H3N2 viruses. 13\n\nA new reassortant genotype of H5N1 containing the HA and NA genes from clade 1.1.2 and the internal genes from clade 2.3.2.1 emerged during 2013 and was associated with the highest number of cases (n = 26) and deaths (n = 14) in Cambodia. 15 Globally since 2003, there have been 650 confirmed H5N1 cases and 386 deaths reported in humans, 16 with most infections in the last 2 years being in children.", "15 Globally since 2003, there have been 650 confirmed H5N1 cases and 386 deaths reported in humans, 16 with most infections in the last 2 years being in children. Human-to-human transmission remains extremely rare based on virological and serological data from analysis of close contacts of confirmed cases in Cambodia, Thailand and Vietnam. 17 A study of household transmission patterns in Indonesia has shown that the overall household attack rate was 18Á3% and the secondary attack rate was 5Á5%, independent of household size.", "17 A study of household transmission patterns in Indonesia has shown that the overall household attack rate was 18Á3% and the secondary attack rate was 5Á5%, independent of household size. 18 Oseltamivir therapy appears to reduce mortality when administered within 8 days of H5N1 illness onset, although earlier treatment is more effective, highlighting the need for early patient diagnosis. 19 Early initiation of oseltamivir was particularly effective in reducing mortality in H5N1 patients without respiratory failure (odds ratio, 0Á17; P = 0Á04), whereas those requiring ventilatory support at the time of oseltamivir initiation were more likely to die.", "19 Early initiation of oseltamivir was particularly effective in reducing mortality in H5N1 patients without respiratory failure (odds ratio, 0Á17; P = 0Á04), whereas those requiring ventilatory support at the time of oseltamivir initiation were more likely to die. 20 A study of the risk factors for mortality related to H5N1 identified age, country, per capita government health expenditure and delay from symptom onset to hospitalisation as the key parameters, highlighting the importance of early diagnosis, treatment and supportive care. 21 High-dose systemic corticosteroids (SC) are associated with worse outcomes in H5N1 patients.", "21 High-dose systemic corticosteroids (SC) are associated with worse outcomes in H5N1 patients. 22 One human case of avian H5N6 was recently detected in China; the virus was a reassortant that contained seven genes from H5N1 and the NA gene from an H6N6 virus circulating in ducks. 23 China has also reported the detection of three human infections, two fatal, with avian H10N8 viruses that contain the internal genes from H9N2, as does H7N9.", "23 China has also reported the detection of three human infections, two fatal, with avian H10N8 viruses that contain the internal genes from H9N2, as does H7N9. 24 Like the H7N9 virus, the H10N8 virus has low pathogenicity in poultry and is therefore difficult to detect in birds. Burden in target populations Pregnant women and infants have an increased risk of complications following influenza infection.", "Burden in target populations Pregnant women and infants have an increased risk of complications following influenza infection. Globally, significant numbers of pregnant women died during the 2009-2010 pandemic, but no maternal mortality occurred in Japan. 25 Through education campaigns directed at pregnant women and healthcare professionals, 67% of pregnant women were vaccinated against H1N1pdm09 resulting in an infection rate among pregnant women in Japan of 3Á5% compared to the overall infection rate in the population of 12%.", "25 Through education campaigns directed at pregnant women and healthcare professionals, 67% of pregnant women were vaccinated against H1N1pdm09 resulting in an infection rate among pregnant women in Japan of 3Á5% compared to the overall infection rate in the population of 12%. 26 Of those pregnant women who were infected with H1N1pdm09 in Japan, 95% were treated with antivirals, and importantly, 88% of those were treated within 2 days of symptom onset. 25 In Mongolia, a prospective cohort study during 2013-2014 found that influenza-like illness (ILI) was detected in 17Á9% of pregnant women, of whom the majority tested positive for influenza A, with substantially lower influenza B and respiratory syncytial virus (RSV) infection.", "25 In Mongolia, a prospective cohort study during 2013-2014 found that influenza-like illness (ILI) was detected in 17Á9% of pregnant women, of whom the majority tested positive for influenza A, with substantially lower influenza B and respiratory syncytial virus (RSV) infection. 27 During the same period, ILI was detected in 30Á9% of infants <6 months of age, with an even spread of influenza A, influenza B and RSV. 27 The influenza burden in children in a rural Indian community was found to be substantial with 11Á6% of ILI cases being caused by influenza A or B viruses.", "27 The influenza burden in children in a rural Indian community was found to be substantial with 11Á6% of ILI cases being caused by influenza A or B viruses. 28 These findings underscore the importance of maternal immunisation. 29 Transmission patterns Sequence analysis of influenza viruses isolated from students on a Singapore University Campus provided insights into the chain of transmission, showing that 62% of 32 viruses were highly similar, demonstrating that the majority of transmission was occurring on the university campus rather than from infections outside.", "29 Transmission patterns Sequence analysis of influenza viruses isolated from students on a Singapore University Campus provided insights into the chain of transmission, showing that 62% of 32 viruses were highly similar, demonstrating that the majority of transmission was occurring on the university campus rather than from infections outside. 30 The effectiveness of surgical masks, hand hygiene and health education investigated in households in Hong Kong and Bangkok detected no significant difference in attack rate in cohorts using one of these interventions. 31 Further analysis of the data enabled some insights into the relative importance of aerosol, large droplet and contact transmission within the households.", "31 Further analysis of the data enabled some insights into the relative importance of aerosol, large droplet and contact transmission within the households. For influenza A infections, aerosol transmission appeared to be the most common route, whereas contact transmission caused the highest number of influenza B infections. 31 \n\nUse and effectiveness Neuraminidase inhibitors are commonly used for the treatment of influenza in Japan, typically following a positive result from a point-of-care (POC) test.", "31 \n\nUse and effectiveness Neuraminidase inhibitors are commonly used for the treatment of influenza in Japan, typically following a positive result from a point-of-care (POC) test. During the 2009-2010 pandemic, over 20 million POC test kits were shipped to hospitals and clinics in Japan to enable rapid diagnosis, and 89% of treated cases were administered NAIs within 48 hours of symptom onset. 32 In Japan during 2013, oseltamivir and laninamivir each represented 40% of NAIs used, while zanamivir (15%) and peramivir (5%) use was considerably less.", "32 In Japan during 2013, oseltamivir and laninamivir each represented 40% of NAIs used, while zanamivir (15%) and peramivir (5%) use was considerably less. NAI effectiveness has been assessed in numerous observational studies in Japan. Oseltamivir effectiveness is significantly reduced in patients with delayed treatment, and duration of fever and viral shedding is longer in treated patients with influenza B compared to influenza A virus infections.", "Oseltamivir effectiveness is significantly reduced in patients with delayed treatment, and duration of fever and viral shedding is longer in treated patients with influenza B compared to influenza A virus infections. 33, 34 The reduced effectiveness against influenza B viruses was also observed in zanamivir 33, 35 and laninamivir 35 trials. To determine whether NAIs reduced mortality during the 2009-2010 pandemic, data were compiled on 29 234 patients hospitalised with confirmed A(H1N1)pdm09 infection.", "To determine whether NAIs reduced mortality during the 2009-2010 pandemic, data were compiled on 29 234 patients hospitalised with confirmed A(H1N1)pdm09 infection. 36 Compared with no treatment, NAI treatment was associated with significantly reduced mortality, with early treatment also showing a reduced risk of mortality compared to late treatment. Although there was no significant clinical effect when comparing late treatment with no treatment in hospitalised patients, there was a significant benefit in treating patients who arrive late into intensive care units.", "Although there was no significant clinical effect when comparing late treatment with no treatment in hospitalised patients, there was a significant benefit in treating patients who arrive late into intensive care units. 36 In a household prophylaxis study, inhaled laninamivir given for either 2 or 3 days reduced the illness rate within households to 3Á9% and 3Á7%, respectively, compared to 16Á9% in households given a placebo. 37 A ferret model of oseltamivir prophylaxis has shown that while morbidity was significantly reduced, the prophylaxis regimes did not prevent infection nor significantly reduce virus load.", "37 A ferret model of oseltamivir prophylaxis has shown that while morbidity was significantly reduced, the prophylaxis regimes did not prevent infection nor significantly reduce virus load. 38 Resistance Although all four NAIs are sialic acid analogues, they have subtle differences in chemical structure and binding properties. Consequently, resistance patterns vary across NAIs.", "Consequently, resistance patterns vary across NAIs. The most commonly detected NA substitution causing NAI resistance in N1-containing influenza viruses is H275Y, which confers resistance to oseltamivir and peramivir, but not to zanamivir and laninamivir. This resistance mutation became fixed in seasonal H1N1 viruses circulating in 2008-2009.", "This resistance mutation became fixed in seasonal H1N1 viruses circulating in 2008-2009. 39 A late 2013 cluster of H1N1pdm09 viruses containing the H275Y substitution was detected in 38 (39%) of 97 H1N1pdm09 viruses from community patients not receiving NAIs in Sapporo, Japan, 40 reminiscent of a similar cluster of oseltamivir-resistant H1N1pdm09 viruses in community patients in Australia in 2011. 41, 42 Importantly, both sets of viruses contained permissive NA mutations (V241I and N369K) that have been shown in ferret studies to offset the destabilising and negative effect of the H275Y NA mutation.", "41, 42 Importantly, both sets of viruses contained permissive NA mutations (V241I and N369K) that have been shown in ferret studies to offset the destabilising and negative effect of the H275Y NA mutation. 43 In hospitalised influenza patients being treated with intravenous zanamivir, next-generation sequencing has been utilised to identify minor resistant virus populations. A total of five NA substitutions were identified in different viruses, including E119K and E119D; however, all apart from E119D were present in such low proportions that they could not be detected by Sanger 'population' sequencing methods.", "A total of five NA substitutions were identified in different viruses, including E119K and E119D; however, all apart from E119D were present in such low proportions that they could not be detected by Sanger 'population' sequencing methods. 44 The effects of various mutations in catalytic and framework residues of influenza B NA were investigated using reverse genetics and a range of functional assays. Four substitutions (D198E, I222T, H274Y and N294S) conferred reduced susceptibility to oseltamivir, while three substitutions (E119A, D198Y and R371K) caused highly reduced inhibition by oseltamivir, zanamivir and peramivir.", "Four substitutions (D198E, I222T, H274Y and N294S) conferred reduced susceptibility to oseltamivir, while three substitutions (E119A, D198Y and R371K) caused highly reduced inhibition by oseltamivir, zanamivir and peramivir. 45 Two of these variants (H274Y, E119A) had in vitro replication fitness comparable to the NAI-susceptible viruses. To date, these substitutions have only been detected on rare occasions in circulating influenza viruses.", "To date, these substitutions have only been detected on rare occasions in circulating influenza viruses. An intravenous formulation of zanamivir showed both virological and clinical effectiveness without safety concerns in patients hospitalised with influenza in Japan. 46 A range of new adamantane derivatives have good antiviral activity in vitro and in animal models against H1N1pdm09 and H3N2 viruses that contain the S31N M2 ion channel substitution that confers resistance to amantadine.", "46 A range of new adamantane derivatives have good antiviral activity in vitro and in animal models against H1N1pdm09 and H3N2 viruses that contain the S31N M2 ion channel substitution that confers resistance to amantadine. 47 Favipiravir is a novel pyrazinamide molecule that inhibits replication of various RNA viruses, including influenza types A, B and C (including oseltamivir-resistant strains), and has recently been licensed in Japan for the control of novel or reemerging influenza viruses. Its triphosphate metabolite is an RNA polymerase inhibitor which disrupts virus genome replication; synergy with oseltamivir has been demonstrated in pre-clinical models.", "Its triphosphate metabolite is an RNA polymerase inhibitor which disrupts virus genome replication; synergy with oseltamivir has been demonstrated in pre-clinical models. 48 A phase II study in the US has shown that a twice-daily regimen decreased the titre and time to cessation of virus shedding, and had a significant benefit in reducing clinical symptoms (NCT01068912; .gov). Subsequent phase III studies are currently ongoing (NCT02008344 and NCT02026349).", "Subsequent phase III studies are currently ongoing (NCT02008344 and NCT02026349). A neutralising monoclonal antibody (MHAA4549A) which binds to the HA stalk of influenza A viruses in both group 1 and group 2 HA subtypes has been effective when given up to 72 hours post-infection in mice and ferrets infected with H5N1. 49 Phase I and IIa trials in humans showed that the antibody was well tolerated, had a mean half-life of 21Á9 days and was effective as therapy at high doses in experimentally infected volunteers (NCT01877785).", "49 Phase I and IIa trials in humans showed that the antibody was well tolerated, had a mean half-life of 21Á9 days and was effective as therapy at high doses in experimentally infected volunteers (NCT01877785). Upcoming placebo-controlled phase IIb trials will target hospitalised influenza patients requiring oxygen and compare the combination of the monoclonal antibody with oseltamivir to oseltamivir monotherapy (NCT01980966). Other broadly neutralising antibodies against multiple clades of H5N1 have been generated by glycan masking of key HA antigenic residues to direct antibody responses to the more conserved stem region of the HA.", "Other broadly neutralising antibodies against multiple clades of H5N1 have been generated by glycan masking of key HA antigenic residues to direct antibody responses to the more conserved stem region of the HA. 50 FluPep, a novel peptide that prevents virus entry into cells, has been shown in mouse studies to be effective in reducing virus titres in lungs, inflammatory cytokines and mortality. 51 Fludase (DAS-181) is a host-targeted therapeutic agent that removes sialic acid from cellular receptors in the respiratory tract, thus preventing influenza virus binding.", "51 Fludase (DAS-181) is a host-targeted therapeutic agent that removes sialic acid from cellular receptors in the respiratory tract, thus preventing influenza virus binding. Delivered topically, it is effective in animal models of lethal H5N1 and H7N9 infection, including a NAI-resistant R292K H7N9 variant. 52 In a phase 2 RCT, inhaled DAS181 reduced pharyngeal viral replication in uncomplicated influenza but did not reduce nasal virus loads or improve clinical outcomes.", "52 In a phase 2 RCT, inhaled DAS181 reduced pharyngeal viral replication in uncomplicated influenza but did not reduce nasal virus loads or improve clinical outcomes. 53 Another receptor-targeted approach is the development of multivalent sialic acid-binding proteins; 54 a single administration 7 days pre-infection resulted in the protection of 80-100% of mice from lethal H7N9 challenge. 55 Apart from blocking sialic acid, the compound appears to stimulate the expression of pro-inflammatory mediators, thereby 'preparing' the immune system for subsequent influenza infection.", "55 Apart from blocking sialic acid, the compound appears to stimulate the expression of pro-inflammatory mediators, thereby 'preparing' the immune system for subsequent influenza infection. When delivered 24 hours post-infection, protection was, however, only 20-40%. 55 Although drug resistance is considered less likely to occur with host-directed therapies, escape mutants have developed rapidly following exposure to a host-directed vacuolar ATPase-inhibiting drug.", "55 Although drug resistance is considered less likely to occur with host-directed therapies, escape mutants have developed rapidly following exposure to a host-directed vacuolar ATPase-inhibiting drug. 56 Furthermore, following serial passage of different viruses in the presence of bafilomycin A1, two HA mutations were selected (A19T and S210N) which resulted in reduced drug susceptibility and increased virulence in mice. 56 \n\nMultiple influenza vaccine effectiveness (IVE) studies have used the control test negative design approach to estimate IVE during early and late phases of influenza seasons, the 2009 pandemic, and by age or target groups.", "56 \n\nMultiple influenza vaccine effectiveness (IVE) studies have used the control test negative design approach to estimate IVE during early and late phases of influenza seasons, the 2009 pandemic, and by age or target groups. Typically, IVE estimates range from 40% to 60% each season. 57 Future studies will investigate IVE with respect to the type of influenza vaccine used, whether IVE differs between the start and end of the season and the effect of previous vaccination.", "57 Future studies will investigate IVE with respect to the type of influenza vaccine used, whether IVE differs between the start and end of the season and the effect of previous vaccination. In Japan in 2013/14, IVE for influenza A in children aged 1-5 years averaged 72% (95% CI 64-79), dropped to 48% (95% CI 31-61) in children aged 6-12 years and was not apparent against influenza B in any age group (À1%, 95% CI À19 to 14). 58 Influenza vaccine effectiveness is known to be lower in adults over 65 years of age, a group that accounts for >60% of seasonal influenza-related hospitalisations and >90% of influenza-related deaths.", "58 Influenza vaccine effectiveness is known to be lower in adults over 65 years of age, a group that accounts for >60% of seasonal influenza-related hospitalisations and >90% of influenza-related deaths. In an effort to improve IVE in the elderly, recent RCTs have investigated the use of adjuvants, intradermal injection and higher doses of antigen. While the use of AS03-adjuvanted influenza vaccine was only moderately superior to non-adjuvanted vaccine in the elderly, 59 the use of a high-dose vaccine containing four times the standard level of HA (60 lg per virus) did result in improved effectiveness compared to the standard dose vaccine.", "While the use of AS03-adjuvanted influenza vaccine was only moderately superior to non-adjuvanted vaccine in the elderly, 59 the use of a high-dose vaccine containing four times the standard level of HA (60 lg per virus) did result in improved effectiveness compared to the standard dose vaccine. 60 For vaccine manufacturers, generating high-growth reassortants of certain circulating viruses can be challenging. A recent study used random mutagenesis of PR8 and selection of high-growth clones in MDCK and Vero cells to derive a high-growth version of PR8.", "A recent study used random mutagenesis of PR8 and selection of high-growth clones in MDCK and Vero cells to derive a high-growth version of PR8. 61 Reassortment of the highgrowth PR8 virus with the HA/NA of either H5N1, H7N9 or seasonal influenza viruses showed that yields significantly exceeded equivalent reassortants that contained the internal genes of the 'normal' PR8 virus. 61 \n\nAs of July 2014, the number of confirmed cases of MERS-CoV has exceeded 830, with at least 288 associated deaths.", "61 \n\nAs of July 2014, the number of confirmed cases of MERS-CoV has exceeded 830, with at least 288 associated deaths. 62 The majority of cases have involved patients with comorbidities (76%) and are predominately males (63%) with a median age of 47. 63, 64 Fewer than 25% of patients have reported contact with animals including dromedary camels, which have been shown to be one likely animal reservoir based on sero-positivity and detection of MERS-CoV.", "63, 64 Fewer than 25% of patients have reported contact with animals including dromedary camels, which have been shown to be one likely animal reservoir based on sero-positivity and detection of MERS-CoV. 65 More than 25% of the infections have been in healthcare workers, and the large number of nosocomial infections is likely due to inadequate infection control in hospitals plus enhanced surveillance that has detected a substantial number of mild or asymptomatic infections. 63 Outside hospital, the burden of disease is likely to be larger than has been reported.", "63 Outside hospital, the burden of disease is likely to be larger than has been reported. 66 Serological analysis of several UK patients found a rapid rise in antibodies from day 10, and that titres were maintained for at least 300 days post-infection. Anti-S (spike glycoprotein) antibodies are responsible for virus neutralisation.", "Anti-S (spike glycoprotein) antibodies are responsible for virus neutralisation. Importantly for serological analyses, patients who experience only mild disease may mount only a modest serological response. 67 Sequential samples from three cases involved in a chain of transmission were extensively analysed using next-generation sequencing.", "67 Sequential samples from three cases involved in a chain of transmission were extensively analysed using next-generation sequencing. 68 Various minority variants were detected, of which some were transient while others were transmitted, and there was evidence of variation in frequency of some variants in different body compartments. Various therapeutic options have been investigated for the treatment of MERS-CoV, but no therapy of proven value currently exists.", "Various therapeutic options have been investigated for the treatment of MERS-CoV, but no therapy of proven value currently exists. The use of SC was associated with adverse outcome in SARS 69 and is not recommended for MERS-CoV. Many agents have shown inhibitory effects against MERS-CoV in cell culture including interferon +/À ribavirin, cyclosporine A, mycophenolic acid, chloroquine and lopinavir.", "Many agents have shown inhibitory effects against MERS-CoV in cell culture including interferon +/À ribavirin, cyclosporine A, mycophenolic acid, chloroquine and lopinavir. 70 Interferons, lopinavir, mycophenolate, possibly alisporivir and combinations are reasonable choices for testing in controlled clinical trials. Exploratory post hoc metaanalysis of studies related to SARS and severe influenza has shown a significant reduction in mortality following convalescent plasma treatment compared to placebo or no therapy (odds ratio 0Á25; 95% CI 0Á14-0Á45).", "Exploratory post hoc metaanalysis of studies related to SARS and severe influenza has shown a significant reduction in mortality following convalescent plasma treatment compared to placebo or no therapy (odds ratio 0Á25; 95% CI 0Á14-0Á45). 71 Thus, the early use of virus-specific neutralising antibodies in the form of convalescent plasma and monoclonal or polyclonal neutralising antibodies for treatment of MERS-CoV has the highest likelihood of clinical benefit. 64 Modalities with risks likely to exceed benefits include SC, ribavirin monotherapy and IVIG.", "64 Modalities with risks likely to exceed benefits include SC, ribavirin monotherapy and IVIG. 72\n\nRespiratory syncytial virus (RSV) Respiratory syncytial virus disproportionately impacts children in low-income countries. 73 Almost all children will have been infected with RSV by their 2nd birthday, and it is the number one cause of hospitalisation of infants in the US, causing 10 times more infant deaths than influenza.", "73 Almost all children will have been infected with RSV by their 2nd birthday, and it is the number one cause of hospitalisation of infants in the US, causing 10 times more infant deaths than influenza. 74 In addition, RSV infects 3-10% of adults annually and accounts for 5-15% of community acquired pneumonia (CAP) and 9-10% of hospitalisations, 75 a burden of disease that approaches that caused by influenza. In a study, conducted in Hong Kong, of 607 hospitalised adults with RSV, 40% had pneumonia and 70% required supplementary oxygen; mortality rates and duration of hospital stay were similar to those observed for influenza patients.", "In a study, conducted in Hong Kong, of 607 hospitalised adults with RSV, 40% had pneumonia and 70% required supplementary oxygen; mortality rates and duration of hospital stay were similar to those observed for influenza patients. 75 Approximately, 15% of hospitalised RSV patients had bacterial superinfections. Although corticosteroids were used to treat 38% of patients, treatment had no benefit on clinical outcome, and instead increased bacterial secondary infections and caused a longer duration of illness.", "Although corticosteroids were used to treat 38% of patients, treatment had no benefit on clinical outcome, and instead increased bacterial secondary infections and caused a longer duration of illness. 75 RSV replication appears prolonged in patients with comorbidities and LRT complications. Palivizumab prophylaxis of premature infants of <6 months of age has been shown to reduce hospitalisation due to RSV by 55%.", "Palivizumab prophylaxis of premature infants of <6 months of age has been shown to reduce hospitalisation due to RSV by 55%. 76 Preventing RSV during infancy has been associated with reduction of wheezing later in life. 77 Trials of other monoclonal antibodies have typically shown that they do not achieve superiority compared to palivizumab and therefore do achieve licensure.", "77 Trials of other monoclonal antibodies have typically shown that they do not achieve superiority compared to palivizumab and therefore do achieve licensure. Furthermore, treatment with neutralising monoclonal antibodies does not appear to reduce virus load or disease severity in hospitalised infants. 78 Alternative options for RSV therapy to be assessed in future clinical trials include inhaled nanobodies, aerosolised peptides, nucleoside analogues and RNA-interference molecules.", "78 Alternative options for RSV therapy to be assessed in future clinical trials include inhaled nanobodies, aerosolised peptides, nucleoside analogues and RNA-interference molecules. Human rhinoviruses (HRV) usually cause mild acute respiratory infections, but on occasions can also cause more severe respiratory infections, including exacerbations of asthma and COPD. Of 115 Japanese children with asthma, a respiratory virus was detected in 86%, of which HRV (n = 36) or RSV (n = 47) were most common.", "Of 115 Japanese children with asthma, a respiratory virus was detected in 86%, of which HRV (n = 36) or RSV (n = 47) were most common. 79 Ex vivo bronchial epithelial cells from people with asthma are more susceptible to HRV infection, due to deficient induction of IFN-b and IFN-lambda. In a study of 147 asthmatics on inhaled corticosteroid therapy, with a history of virusassociated exacerbations, patients were randomised to 14-day treatment with inhaled IFN-b or placebo within 24 hours of developing cold symptoms.", "In a study of 147 asthmatics on inhaled corticosteroid therapy, with a history of virusassociated exacerbations, patients were randomised to 14-day treatment with inhaled IFN-b or placebo within 24 hours of developing cold symptoms. Patients who received IFN-b had enhanced morning peak expiratory flow recovery, reduced need for additional treatment and boosted innate immunity as assessed by blood and sputum biomarkers. In an exploratory analysis of a subset of more difficult-to-treat asthma (n = 27 IFN-b; n = 31 placebo), worsening of symptoms increased significantly in the placebo group, but was prevented by IFN-b (P = 0Á004).", "In an exploratory analysis of a subset of more difficult-to-treat asthma (n = 27 IFN-b; n = 31 placebo), worsening of symptoms increased significantly in the placebo group, but was prevented by IFN-b (P = 0Á004). 80 A picornavirus-specific antiviral, vapendavir, was found to reduce symptom scores, lower bronchodilator puffer use and reduce viral load in asthma patients with an URTI due to HRV. 81 \n\nDiagnostics Point-of-care tests that can deliver a result in 15 minutes have been available in many countries for the last decade, but while having good specificity, the sensitivity has typically been poor, ranging from 10% to 80% compared to PCR or culture.", "81 \n\nDiagnostics Point-of-care tests that can deliver a result in 15 minutes have been available in many countries for the last decade, but while having good specificity, the sensitivity has typically been poor, ranging from 10% to 80% compared to PCR or culture. Their use in emergency departments of hospitals can result in reduced unnecessary antibiotic use and an increased likelihood of discharge. Newer immunofluorescence-based POC tests with improved sensitivity are being developed.", "Newer immunofluorescence-based POC tests with improved sensitivity are being developed. In addition, the Quidel Sofia POC test may be linked via the internet such that results can be reported in real-time to central databases. Other POC tests are using photographic silver amplification immunochromatography technology to increase sensitivity.", "Other POC tests are using photographic silver amplification immunochromatography technology to increase sensitivity. 82 PCR remains the gold standard for virus diagnostics with an ability to be rapid, sensitive, specific and to identify a wide range of pathogens via different assays. The ability to multiplex multiple pathogen targets allows costs to be reduced in a diagnostic setting.", "The ability to multiplex multiple pathogen targets allows costs to be reduced in a diagnostic setting. New closed-system technologies which involve only minimal hands-on time (a few minutes) and that conduct both automated nucleic acid extraction and PCR for multiple pathogens are now available, but are currently limited for clinical diagnostic purposes due to low-throughput capabilities. 83 Next-generation sequencing technologies and PCR-based analyses with increasing sensitivity both offer considerable scope in diagnosis, although our current understanding of the clinical impact of pathogens at low levels or the presence of variants as minor virus populations is limited.", "83 Next-generation sequencing technologies and PCR-based analyses with increasing sensitivity both offer considerable scope in diagnosis, although our current understanding of the clinical impact of pathogens at low levels or the presence of variants as minor virus populations is limited. Providing low-cost, sensitive assays for diagnosing respiratory virus infections in low/middle-income countries is challenging, but has the potential to improve treatment and avoid unnecessary antibiotic use in these regions. Repurposed drugs for respiratory viral infections Nitazoxanide (NTX) is an antiparasitic agent approved for Giardia and Cryptosporidium infections that also inhibits replication in vitro of influenza and other respiratory viruses.", "Repurposed drugs for respiratory viral infections Nitazoxanide (NTX) is an antiparasitic agent approved for Giardia and Cryptosporidium infections that also inhibits replication in vitro of influenza and other respiratory viruses. 84 Treatment with NTX 600 mg twice daily for 5 days was associated with a reduction in the duration of symptoms in participants with acute uncomplicated influenza. 85 In a subset analysis of 238 patients with no confirmed virus infection, treatment with NTX 600 mg also led to a shorter time to alleviation of symptoms in comparison to placebo (88Á4 versus 105Á7 hours, P = 0Á02).", "85 In a subset analysis of 238 patients with no confirmed virus infection, treatment with NTX 600 mg also led to a shorter time to alleviation of symptoms in comparison to placebo (88Á4 versus 105Á7 hours, P = 0Á02). 86 Systemic corticosteroids for respiratory virus infections A review of prospective observational studies has shown that SC increased the risks of mortality and morbidity (e.g. secondary infections, hospital-acquired pneumonia) in severe infection due to influenza A(H1N1)pdm09 especially with delayed antiviral therapy.", "secondary infections, hospital-acquired pneumonia) in severe infection due to influenza A(H1N1)pdm09 especially with delayed antiviral therapy. 87 During SARS infections, a higher risk of avascular necrosis and prolonged virus shedding were observed in patients who had received highdose SC therapy. 88 It is therefore important to avoid the use of high-dose SC in severe respiratory viral infections outside the context of clinical trials.", "88 It is therefore important to avoid the use of high-dose SC in severe respiratory viral infections outside the context of clinical trials. Larger trials are needed to resolve the uncertainty regarding the effect of early SC therapy in ARDS. Low-dose SC is indicated for management of refractory septic shock, 89 and a short course of SC is indicated for acute exacerbations of obstructive airway diseases (asthma, COPD) 90 Current evidence does not support a clinically relevant effect of systemic or inhaled glucocorticoids on admission or length of hospitalisation for acute viral bronchiolitis in infants and young children.", "Low-dose SC is indicated for management of refractory septic shock, 89 and a short course of SC is indicated for acute exacerbations of obstructive airway diseases (asthma, COPD) 90 Current evidence does not support a clinically relevant effect of systemic or inhaled glucocorticoids on admission or length of hospitalisation for acute viral bronchiolitis in infants and young children. 91 \n\nRespiratory syncytial virus, influenza viruses, parainfluenza (PIV) viruses and adenoviruses (AdVs) cause the most serious disease in immunocompromised hosts, but other respiratory viruses are becoming increasingly appreciated as a cause of both upper and lower respiratory tract disease. The potential for these viruses to cause lower respiratory tract infections (LRTI) after transplantation varies.", "The potential for these viruses to cause lower respiratory tract infections (LRTI) after transplantation varies. Human metapneumovirus infections have similar outcomes to RSV infection in hematopoietic stem cell transplant (HSCT) recipients, including potentially severe and fatal pneumonia. HRV and coronavirus infections are very frequent in transplant recipients, but severe lower respiratory tract disease is uncommon.", "HRV and coronavirus infections are very frequent in transplant recipients, but severe lower respiratory tract disease is uncommon. In a prospective study of 112 lung transplant recipients, the virus infection rates upon screening, routine and emergency visits were 14%, 15% and 34%, respectively. Picornaviruses were identified most frequently in nasopharyngeal (85/140; 61%) and BAL specimens (20/34; 59%).", "Picornaviruses were identified most frequently in nasopharyngeal (85/140; 61%) and BAL specimens (20/34; 59%). Asymptomatic virus carriage, mainly of picornaviruses, was found at 10% of screening visits. Infections were associated with transient lung function loss and high calcineurin inhibitor blood levels.", "Infections were associated with transient lung function loss and high calcineurin inhibitor blood levels. The hospitalisation rate was 50% for influenza and PIV and 16Á9% for other viruses. Acute rejection was not associated with virus infection.", "Acute rejection was not associated with virus infection. 92 The risk factors for severe LRTI among transplant recipients include early onset post-transplant (<3 m), steroid boluses, young children (<1 year), chronic GVHD, lymphopenia/lymphodepletion and allogeneic HSCT patients. 92 Influenza Immunocompromised patients with influenza exhibit more complications, longer virus shedding and more antiviral resistance, while often demonstrating milder clinical symptoms and signs on initial clinical assessment.", "92 Influenza Immunocompromised patients with influenza exhibit more complications, longer virus shedding and more antiviral resistance, while often demonstrating milder clinical symptoms and signs on initial clinical assessment. 93 Influenza A (H1N1)pdm09 viruses have the potential for rapid emergence of oseltamivir resistance and causing severe morbidity, particularly in immunocompromised patients with lymphopenia and delayed antiviral therapy. 94 Influenza viraemia may serve as a marker for overall poor outcome with increased risk of progression to LRTI, hypoxaemia, respiratory failure and death.", "94 Influenza viraemia may serve as a marker for overall poor outcome with increased risk of progression to LRTI, hypoxaemia, respiratory failure and death. Influenza RNA in blood (viraemia) was detected in nine of 79 (11Á4%) HSCT recipients with influenza. Among patients with LRTI, viraemia was associated with increased hazards of overall as well as influenzaassociated death (hazard ratio 3Á5, 1Á1-12).", "Among patients with LRTI, viraemia was associated with increased hazards of overall as well as influenzaassociated death (hazard ratio 3Á5, 1Á1-12). 95 In 143 HSCT recipients with documented seasonal influenza infection, treatment with high-dose corticosteroids was associated with a trend towards prolonged virus shedding [(OR), 3Á3; 95% CI 1Á0-11; P = 0Á05], whereas antiviral therapy initiated to treat upper respiratory tract infection (URTI) was associated with fewer cases of LRTI (OR, 0Á04; 95% CI, 0-0Á2; P < 0Á01) and fewer hypoxaemia episodes (OR, 0Á3; 95% CI, 0Á1-0Á9; P = 0Á03). 96 In view of the risks of prolonged replication and drug resistance emergence, 97 a longer duration and a higher NAI dose may be beneficial.", "96 In view of the risks of prolonged replication and drug resistance emergence, 97 a longer duration and a higher NAI dose may be beneficial. Early therapy was consistently demonstrated to have improved outcomes. [98] [99] [100] [101] Other treatment options under study are the use of triple combination therapy with amantadine, oseltamivir and ribavirin, 102 or intravenous peramivir 103 or zanamivir.", "[98] [99] [100] [101] Other treatment options under study are the use of triple combination therapy with amantadine, oseltamivir and ribavirin, 102 or intravenous peramivir 103 or zanamivir. 104 Therapy of influenza in lung transplant recipients is associated with a reduced risk of developing bronchiolitis obliterans syndrome. 105 Parainfluenza DAS181 is inhibitory for PIV and influenza viruses, including those resistant to the amantadine and NAIs 106 and may be effective in treating immunocompromised patients with severe PIV lung disease.", "105 Parainfluenza DAS181 is inhibitory for PIV and influenza viruses, including those resistant to the amantadine and NAIs 106 and may be effective in treating immunocompromised patients with severe PIV lung disease. 107 In a study of four severely immunocompromised children with PIV disease treatment with DAS181 for 5-10 days, by dry powder inhalation or nebulisation, was well tolerated. Transient increase in serum alkaline phosphatase, liver function and coagulation tests were observed, but nasal wash virus loads were reduced in all patients within 1 week with improved clinical features.", "Transient increase in serum alkaline phosphatase, liver function and coagulation tests were observed, but nasal wash virus loads were reduced in all patients within 1 week with improved clinical features. 108 \n\nIn a RCT of lung transplant recipients with RSV infection, the incidence of new or progressive bronchiolitis obliterans syndrome at day 90 was significantly reduced in 16 patients who received a small interfering RNA against the RSV Ngene (ALN-RSV01) compared with placebo (n = 8) (6Á3% versus 50%, P = 0Á027). 109 In a larger follow-up multicentre phase IIb study, treatment with ALN-RSV01 showed a greater than eightfold reduced risk in developing bronchiolitis obliterans syndrome at day 180.", "109 In a larger follow-up multicentre phase IIb study, treatment with ALN-RSV01 showed a greater than eightfold reduced risk in developing bronchiolitis obliterans syndrome at day 180. 110\n\nAdenovirus is a serious, often fatal infection in immunocompromised patients, especially in HSCT recipients. The control of AdV is mostly T-cell mediated, and therefore, patients who have received T-cell suppressive regimens are at an increased risk for AdV infection.", "The control of AdV is mostly T-cell mediated, and therefore, patients who have received T-cell suppressive regimens are at an increased risk for AdV infection. The annual incidence of AdV infections in HSCT recipients ranges from 5% to 50%, and is increasing, likely due to increased use of T-celldepleted allografts and cord blood as source. The mortality rate is up to 80%.", "The mortality rate is up to 80%. 111 Brincidofovir (BCV; formerly CMX-001) is an orally bioavailable lipid-conjugate of cidofovir (CDV) that provides high intracellular concentrations of CDV diphosphate with a long intracellular half-life (up to 4-6Á5 days). BCV is 65-fold more potent against AdV than CDV in vitro with a low risk of myeloidor nephrotoxicity, but gastrointestinal side effects are more common.", "BCV is 65-fold more potent against AdV than CDV in vitro with a low risk of myeloidor nephrotoxicity, but gastrointestinal side effects are more common. 112 In a retrospective study of 13 immunocompromised patients given BCV for AdV disease after failing or intolerance to i.v. cidofovire nine patients (69Á2%) demonstrated a virological response (VR), which was defined as a 99% drop from baseline or undetectable AdV DNA in serum by week 8.", "cidofovire nine patients (69Á2%) demonstrated a virological response (VR), which was defined as a 99% drop from baseline or undetectable AdV DNA in serum by week 8. Patients with VR had longer survival than those without VR (median 196 days versus 54Á5 days; P = 0Á04). 113" ]

[ "The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients).", "Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage.", "Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites.", "Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread.", "Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration.", "The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] .", "Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] .", "Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms.", "In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital.", "This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines.", "Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube.", "In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s).", "Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government.", "To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included.", "In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001).", "The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded.", "Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC.", "He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8.", "In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again.", "On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation.", "Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38).", "The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample.", "Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation.", "He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13.", "Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation.", "Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out.", "On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days.", "Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU.", "Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.", "In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001).", "In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness.", "Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102).", "In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.", "We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication.", "Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day.", "For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] .", "Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.", "We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] .", "Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.", "Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol.", "Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction." ]

[ "The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients).", "Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage.", "Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites.", "Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread.", "Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration.", "The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] .", "Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] .", "Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms.", "In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital.", "This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines.", "Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube.", "In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s).", "Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government.", "To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included.", "In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001).", "The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded.", "Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC.", "He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8.", "In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again.", "On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation.", "Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38).", "The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample.", "Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation.", "He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13.", "Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation.", "Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out.", "On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days.", "Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU.", "Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.", "In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001).", "In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness.", "Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102).", "In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.", "We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication.", "Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day.", "For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] .", "Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.", "We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] .", "Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.", "Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol.", "Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction." ]

[ "The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients).", "Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage.", "Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites.", "Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread.", "Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration.", "The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] .", "Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] .", "Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms.", "In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital.", "This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines.", "Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube.", "In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s).", "Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government.", "To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included.", "In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001).", "The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded.", "Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC.", "He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8.", "In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again.", "On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation.", "Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38).", "The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample.", "Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation.", "He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13.", "Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation.", "Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out.", "On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days.", "Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU.", "Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.", "In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001).", "In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness.", "Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102).", "In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.", "We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication.", "Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day.", "For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] .", "Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.", "We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] .", "Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.", "Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol.", "Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction." ]

[ "The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients).", "Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage.", "Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites.", "Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread.", "Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration.", "The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] .", "Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] .", "Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms.", "In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital.", "This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines.", "Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube.", "In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s).", "Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government.", "To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included.", "In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001).", "The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded.", "Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC.", "He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8.", "In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again.", "On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation.", "Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38).", "The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample.", "Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation.", "He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13.", "Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation.", "Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out.", "On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days.", "Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU.", "Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.", "In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001).", "In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness.", "Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102).", "In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.", "We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication.", "Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day.", "For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] .", "Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.", "We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] .", "Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.", "Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol.", "Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction." ]

[ "The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients).", "Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage.", "Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites.", "Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread.", "Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration.", "The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] .", "Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] .", "Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms.", "In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital.", "This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines.", "Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube.", "In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s).", "Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government.", "To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included.", "In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001).", "The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded.", "Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC.", "He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8.", "In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again.", "On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation.", "Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38).", "The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample.", "Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation.", "He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13.", "Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation.", "Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out.", "On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days.", "Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU.", "Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.", "In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001).", "In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness.", "Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102).", "In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.", "We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication.", "Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day.", "For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] .", "Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.", "We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] .", "Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.", "Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol.", "Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction." ]

[ "The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients).", "Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage.", "Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites.", "Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread.", "Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration.", "The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] .", "Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] .", "Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms.", "In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital.", "This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines.", "Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube.", "In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s).", "Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government.", "To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included.", "In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001).", "The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded.", "Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC.", "He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8.", "In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again.", "On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation.", "Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38).", "The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample.", "Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation.", "He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13.", "Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation.", "Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out.", "On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days.", "Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU.", "Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.", "In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001).", "In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness.", "Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102).", "In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.", "We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication.", "Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day.", "For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] .", "Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.", "We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] .", "Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.", "Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol.", "Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction." ]

[ "The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients).", "Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage.", "Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites.", "Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread.", "Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration.", "The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] .", "Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] .", "Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms.", "In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital.", "This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines.", "Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube.", "In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s).", "Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government.", "To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included.", "In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001).", "The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded.", "Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC.", "He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8.", "In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again.", "On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation.", "Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38).", "The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample.", "Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation.", "He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13.", "Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation.", "Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out.", "On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days.", "Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU.", "Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.", "In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001).", "In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness.", "Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102).", "In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.", "We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication.", "Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day.", "For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] .", "Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.", "We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] .", "Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.", "Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol.", "Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction." ]

[ "The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients).", "Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage.", "Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites.", "Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread.", "Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration.", "The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] .", "Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] .", "Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms.", "In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital.", "This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines.", "Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube.", "In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s).", "Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government.", "To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included.", "In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001).", "The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded.", "Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC.", "He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8.", "In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again.", "On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation.", "Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38).", "The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample.", "Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation.", "He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13.", "Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation.", "Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out.", "On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days.", "Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU.", "Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.", "In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001).", "In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness.", "Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102).", "In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.", "We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication.", "Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day.", "For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] .", "Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.", "We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] .", "Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.", "Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol.", "Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction." ]

[ "The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients).", "Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage.", "Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites.", "Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread.", "Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration.", "The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] .", "Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] .", "Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms.", "In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital.", "This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines.", "Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube.", "In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s).", "Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government.", "To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included.", "In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001).", "The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded.", "Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC.", "He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8.", "In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again.", "On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation.", "Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38).", "The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample.", "Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation.", "He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13.", "Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation.", "Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out.", "On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days.", "Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU.", "Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.", "In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001).", "In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness.", "Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102).", "In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.", "We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication.", "Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day.", "For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] .", "Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.", "We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] .", "Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.", "Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol.", "Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction." ]

[ "The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients).", "Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage.", "Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites.", "Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread.", "Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration.", "The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] .", "Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] .", "Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms.", "In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital.", "This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines.", "Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube.", "In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s).", "Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government.", "To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included.", "In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001).", "The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded.", "Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC.", "He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8.", "In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again.", "On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation.", "Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38).", "The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample.", "Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation.", "He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13.", "Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation.", "Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out.", "On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days.", "Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU.", "Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.", "In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001).", "In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness.", "Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102).", "In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.", "We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication.", "Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day.", "For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] .", "Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.", "We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] .", "Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.", "Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol.", "Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction." ]

[ "Background: The Ebola epidemic in West Africa caused global fear and stirred up worldwide preparedness activities in countries sharing borders with those affected, and in geographically far-away countries such as Iceland. Objective: To describe and analyse Ebola preparedness activities within the Icelandic healthcare system, and to explore the perspectives and experiences of managers and frontline health workers. Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team.", "Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team. Results: Contextual factors such as culture and demography influenced preparedness, and contributed to the positive state of mind of participants, and ingenuity in using available resources for preparedness. While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland.", "While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families. There were also concerns about the teamwork and lack of commitment by some during training.", "There were also concerns about the teamwork and lack of commitment by some during training. Being a ‘tiny’ nation was seen as both an asset and a weakness in the preparation process. Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans.", "Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so. For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support.", "For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support. Text: Global health; prevention and control; public policy; qualitative evaluation; emergency responders; communicable diseases; emerging; fear Background On 8 August 2014, the World Health Organization declared the Ebola epidemic in West Africa as a Public Health Emergency of International Concern (PHEIC) under the International Health Regulations (IHR) [1] . All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems [2] [3] [4] .", "All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems [2] [3] [4] . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola [5] , there was a growing fear of transmission beyond the region. In breach of WHO recommendations and guidelines [6] , flights were cancelled and cross-border movement curtailed [7] .", "In breach of WHO recommendations and guidelines [6] , flights were cancelled and cross-border movement curtailed [7] . The epidemic caused public concern outside West Africa [8] , as fear and racism found fertile ground [9] [10] [11] , and in an effort to stop the international spread of the disease, all states were advised to be prepared to detect, investigate, and manage Ebola cases [1] . Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' [12] .", "Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' [12] . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied [13] . An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A (H1N1) pandemic revealed that the way plans were framed varied considerably, and '[told] us something about how the different countries want pandemics and preparedness to be understood by the public' [14] .", "An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A (H1N1) pandemic revealed that the way plans were framed varied considerably, and '[told] us something about how the different countries want pandemics and preparedness to be understood by the public' [14] . More research was encouraged into cultural and social structures in the respective countries. In Iceland, information about the Ebola epidemic in West Africa came from several sources.", "In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health (DH) first reported on the epidemic on 8 April 2014 [15] . In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered.", "In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered. Fear of a global epidemic was rife, and in media and online discussions, doubts were raised about the Icelandic health system´s capacity to take care of a patient with Ebola [16] [17] [18] , despite its ranking as one of the best in the world [16] . On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola [19] .", "On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola [19] . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital (LSH), the Department of Civil Protection and Emergency Management (DCPEM), and the seven Primary Healthcare Regional Organizations in the country at the time. Key external partners were the European Centre for Disease Prevention and Control (ECDC) and WHO, in addition to Nordic collaborators in epidemic preparedness [20] .", "Key external partners were the European Centre for Disease Prevention and Control (ECDC) and WHO, in addition to Nordic collaborators in epidemic preparedness [20] . At the same time, it was regarded as highly unlikely that Ebola Virus Disease (EVD) would spread in the country [21] . Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities [22] [23] [24] [25] .", "Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities [22] [23] [24] [25] . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa. Parked in a closed-off area, a physician in full Personal Protective Equipment (PPE) entered the plane, but quickly ruled out Ebola [26] .", "Parked in a closed-off area, a physician in full Personal Protective Equipment (PPE) entered the plane, but quickly ruled out Ebola [26] . Irrespective of good or bad overall performance, health systems are tested in times of crisis, such as epidemics. Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process.", "Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland [16, 27] . Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training.", "Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training. The study was conducted in April-May 2016, and the interviewed participants were administrators and frontline health professionals central to the case, so as to explore their perspectives and experiences concerning Ebola preparedness [28, 29] . Staff in managerial positions were contacted by one of the authors (GG) for permission to interview them based on their role in the preparedness plan.", "Staff in managerial positions were contacted by one of the authors (GG) for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team (ETT), the director of the team listed relevant email contacts. Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors (ÍEH), a physiotherapist.", "Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors (ÍEH), a physiotherapist. In case interviewees suggested other potential participants, they were invited through email to participate. A similar methodology was applied to identify participants from the Emergency Room (ER).", "A similar methodology was applied to identify participants from the Emergency Room (ER). They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness. Three separate interview guides were developedone each for managers, ETT, and ER respectively (see supplementary material).", "Three separate interview guides were developedone each for managers, ETT, and ER respectively (see supplementary material). The interviews included open questions probing the role of their institution in preparedness, the experience of the training process, challenges encountered or expected, and any dilemmas that they may have experienced in relation to the preparedness plan. The recruitment of participants was concluded when saturation was reached.", "The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis. The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views.", "The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views. From this exercise, three broad themes were inductively identified that corresponded to critical perspectives introduced by the participants. Permission to conduct the study was granted by Iceland's National Bioethics Committee (VSN- and Landspitali University Hospital (LSH 13-16, 4 February 2016) .", "Permission to conduct the study was granted by Iceland's National Bioethics Committee (VSN- and Landspitali University Hospital (LSH 13-16, 4 February 2016) . Reporting on the results was guided by the COREC guidelines [30] ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations. The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH [31] , and the preparatory activities engaged more than two hundred staff across all of its departments.", "The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH [31] , and the preparatory activities engaged more than two hundred staff across all of its departments. The ETT consisted of about 50 healthcare professionals who had volunteered to participate, including 11 doctors and 28 nurses, a few laboratory technicians, radiologists, and auxiliary nurses. They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care.", "They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD [31] . Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan.", "Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan. One manager concluded:\n\nYou get no discount. You can never go the shorter way. There was always something that surprised you.", "You can never go the shorter way. There was always something that surprised you. We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems.", "We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems. The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'.", "Ebola preparedness was 'a job that had to be done', and 'someone had to do it'. Some referred to ethical or professional obligations: This is just a part of being a nurse, to encounter situations that can be dangerous to you or someone else, but you have made this decision and you deal with it. Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV.", "Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV. Yet, the practice of volunteering in the preparation was questioned. One participant said:\n\nWe learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with.", "One participant said:\n\nWe learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with. ER staff indicated that for them working in the ER was enough of a risk to take, no reason to expose oneself even more by joining the ETT, and appreciated that others had volunteered. All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways.", "All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways. Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear.", "Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear. Those in managerial positions had listened and taken their opinions into consideration. One said:\n\nThey were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were.", "One said:\n\nThey were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were. Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan.", "Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan. Facilities for isolation and treatment of patients with Ebola were less than perfect:\n\nWe assessed how we could use the department … and change it in just a few hours into some kind of an isolation unit that we could possibly use. Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere.", "Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere. There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory.", "Respondents believed that the training and education of the members of the ETT and ER had been satisfactory. They felt that it had been proportionate to the risk, while some were concerned about the lack of staff. Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment.", "Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment. Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as [we] could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients?", "There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients? When asked if Ebola was a real threat to the country, participants usually referred to how easy it was to travel the globe: 'Yeah, why not, the world is getting smaller'. Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland.", "Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland. One respondent said:\n\nPeople were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts.", "Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness [would have been] needed in the PPE practices'. It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT.", "It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT. When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE.", "Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said:\n\nWhat bothered most of us was how uncomfortable the PPE was and I think that made people nervous: \"How will I manage working in this for hours?\" Another described the donning/doffing process like a 'complicated ballroom dance'.", "Another described the donning/doffing process like a 'complicated ballroom dance'. Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these [non-clinical] people belonged in the team, because this is a very clinical environment in addition to having to be in this costume [PPE] with the risk of becoming infected by mistake.", "One participant said: I didn't think these [non-clinical] people belonged in the team, because this is a very clinical environment in addition to having to be in this costume [PPE] with the risk of becoming infected by mistake. Those with non-clinical background were, however, aware of their limitations: I realized that I would not be the one in the front, I would not be managing patients directly. The importance ascribed to teamwork was evident in relation to fear.", "The importance ascribed to teamwork was evident in relation to fear. Participants described fear of working with people they had not worked with before:\n\nThe weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, [we] help each other.", "Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, [we] help each other. Then you don't want to be working with someone who didn't show up. There were a lot of doctors who just dropped in, dropped out, and then dropped in again.", "There were a lot of doctors who just dropped in, dropped out, and then dropped in again. I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland.", "Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland. A major precaution was planning to avoid contact with the family while working with Ebola patients. One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew (s)he was working with an Ebola patient.", "One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew (s)he was working with an Ebola patient. For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team.", "ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team. 'The normal insurance for LHS staff would not cover everything if we were to become sick or even lose our lives.' Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT.", "Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT. One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need.", "Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need. Characteristics associated with Iceland and the Icelandic people were referred to repeatedly by participants. The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations.", "The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations. 'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made.", "even though compromises had to be made. Comparing how Iceland handled its responsibilities differently from other countries of a larger size was often brought up, both with pride in Iceland as a strong independent nation, and with insecurities about its capacity in comparison to other countries. It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role.", "It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role. As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors.", "Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors. Notions of Icelanders as resilient in spite of shortcomings were common; referring to the experience of preparedness planning and training, one health staff said:\n\nIt was very much the Icelandic way, we'll manage, we'll work it out, and there was so much ingenuity. This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement:\n\nWould it have worked?", "This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement:\n\nWould it have worked? Yes, it would have worked. Would it have been optimal?", "Yes, it would have worked. Would it have been optimal? We cannot say, it would have been optimal; we can say, it would have been sufficient.", "We cannot say, it would have been optimal; we can say, it would have been sufficient. In contrast to this, there were concerns about whether Icelandic aid workers falling ill in Ebolaaffected countries should be transferred to Iceland or to hospitals in other Nordic countries with better isolation units. Some of the participants trusted that patients with EVD would not be transferred to Iceland.", "Some of the participants trusted that patients with EVD would not be transferred to Iceland. One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country.", "We don't know what would have happened if they would have transferred an Icelander into the country. We don't have good enough isolation unitsyou are not supposed to send patients to a hospital that is less than 100%. I thought there was assurance in that.", "I thought there was assurance in that. During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe [32] , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic [33] , in the EU region [34, 35] , and the US [36] .", "Evaluations have, for example, been conducted in 11 African countries close to the epidemic [33] , in the EU region [34, 35] , and the US [36] . Here we present data from a qualitative case study on the process, and experiences with establishing a preparedness plan for Ebola in Iceland in 2014. Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response.", "Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response. The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' (theme 1) refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation.", "'Getting the job done' (theme 1) refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation. Using the metaphors of 'hard core' and 'soft periphery', Langley and Denis [37] emphasize the importance of relatively 'peripheral' concerns and processes for planning and implementation of new interventions. The hard core represents the actual intervention or goal, e.g.", "The hard core represents the actual intervention or goal, e.g. implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core.", "The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core. If the soft periphery is neglected, it will cause multiple challenges in the implementation process, and the benefit of the hard core, the intervention itself, may not transpire as anticipated. Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders.", "Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders. In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan.", "The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan. Comprehensive consultations and the involvement of many associated institutions were mentioned. Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g.", "Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g. when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' [38] .", "During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' [38] . The West African Ebola epidemic was covered widely by media [39] , and the fear of Ebola reached every corner of the world, exemplified by travel bans from affected countries, and trade barriers [40] , in contrast to the ongoing epidemic in the Democratic Republic of Congo [41, 42] . In our second theme, trust, doubt, and fear of health workers were represented.", "In our second theme, trust, doubt, and fear of health workers were represented. Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned.", "The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned. In preparedness efforts in other countries, scarcity of resources in relation to manpower demand and problems with training and protocols involving PPE were common challenges [35] . Similar problems were encountered in Iceland.", "Similar problems were encountered in Iceland. Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere [43] . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal.", "Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal. The members of the ETT pointed out that they had never worked together as a team under circumstances that demanded strict adherence to infectious control procedures. This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation [44] .", "This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation [44] . Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics [35] . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada [45, 46] .", "This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada [45, 46] . Anthropological work on emergency preparedness emphasizes the connectedness between health professionals, technological devices, and knowledge as a prerequisite for successful preparedness. Wolf and Hall present preparedness efforts as a form of governance that involves human bodies (those of health professionals), clinical architectures (e.g.", "Wolf and Hall present preparedness efforts as a form of governance that involves human bodies (those of health professionals), clinical architectures (e.g. isolation wards), and technical artefacts (gloves, protective suits, disinfectants, etc.) [47] .", "[47] . During preparedness training and implementation, 'nursing bodies are transformed into instruments of preparedness', and become part of infrastructural arrangements. Health professionals are, here, both vulnerable and powerful tools in the management of contamination.", "Health professionals are, here, both vulnerable and powerful tools in the management of contamination. The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation.", "In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation. Repeated enactment of receiving and treating a patient with Ebola within experienced and trustful teams would probably enhance such embodiment, provided that there is justified trust in the involved technology. In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested.", "In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested. In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland [48] .", "Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland [48] . Notions of the intrinsic character of Iceland, and of being Icelandic, serve to underscore certain points and explain positive and negative experiences with the preparedness plan. Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere.", "Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere. In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' (things always have a way of working out in the end). The preparedness plan had to be completed, and adapted to Iceland's particular global situation.", "The preparedness plan had to be completed, and adapted to Iceland's particular global situation. In the 21st century, the world has faced new epidemic threats, such as SARS, and old scourges such as the plague have resurfaced [38] . One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 [35] .", "One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 [35] . Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 [49] .", "A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 [49] . During the elaboration of these plans, communication among the different levels of the healthcare system and supporting agencies, such as the DCPEM, had been clearly defined, and proved to be useful in the preparedness for Ebola. Further, as found important in preparedness activities for pandemic influenzae elsewhere [44] , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical.", "Further, as found important in preparedness activities for pandemic influenzae elsewhere [44] , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical. It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics [38] . Iceland was far away from the epicentre of the Ebola epidemic in West Africa.", "Iceland was far away from the epicentre of the Ebola epidemic in West Africa. Yet this case study shows that health professionals felt the strain of possibly having to treat one or more patients with EVD. Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD.", "Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD. Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process.", "References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process. These references negotiate the role and character of the nation of Iceland, and its role in a globalized world, as both a small and isolated nation on one hand, and a central and capable one, on the other. The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination.", "The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination. This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola (or other contagious global health threats) as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline.", "This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline. Geir Gunnlaugsson conceptualized the study, and took part in all necessary steps towards its completion, such as analysis and interpretation of data, and writing the manuscript for submission. Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version.", "Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version. Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version.", "Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version. Dr. Gunnlaugsson reports he was the Chief Medical Officer (CMO) for Iceland, Directorate of Health, in the period 2010-2014. Other authors report no conflict of interest.", "Other authors report no conflict of interest. The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland (number VSI- ). Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 (number LSH [13] [14] [15] [16] .", "Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 (number LSH [13] [14] [15] [16] . Participants signed an informed consent form before taking part in the study. Not applicable.", "Participants signed an informed consent form before taking part in the study. Not applicable. The manuscript builds on the work of Íris Eva Hauksdóttir towards a MSc in Global Health, Section of Global Health, Department of Public Health, Copenhagen University, Denmark." ]

[ "Background: The Ebola epidemic in West Africa caused global fear and stirred up worldwide preparedness activities in countries sharing borders with those affected, and in geographically far-away countries such as Iceland. Objective: To describe and analyse Ebola preparedness activities within the Icelandic healthcare system, and to explore the perspectives and experiences of managers and frontline health workers. Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team.", "Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team. Results: Contextual factors such as culture and demography influenced preparedness, and contributed to the positive state of mind of participants, and ingenuity in using available resources for preparedness. While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland.", "While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families. There were also concerns about the teamwork and lack of commitment by some during training.", "There were also concerns about the teamwork and lack of commitment by some during training. Being a ‘tiny’ nation was seen as both an asset and a weakness in the preparation process. Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans.", "Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so. For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support.", "For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support. Text: Global health; prevention and control; public policy; qualitative evaluation; emergency responders; communicable diseases; emerging; fear Background On 8 August 2014, the World Health Organization declared the Ebola epidemic in West Africa as a Public Health Emergency of International Concern (PHEIC) under the International Health Regulations (IHR) [1] . All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems [2] [3] [4] .", "All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems [2] [3] [4] . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola [5] , there was a growing fear of transmission beyond the region. In breach of WHO recommendations and guidelines [6] , flights were cancelled and cross-border movement curtailed [7] .", "In breach of WHO recommendations and guidelines [6] , flights were cancelled and cross-border movement curtailed [7] . The epidemic caused public concern outside West Africa [8] , as fear and racism found fertile ground [9] [10] [11] , and in an effort to stop the international spread of the disease, all states were advised to be prepared to detect, investigate, and manage Ebola cases [1] . Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' [12] .", "Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' [12] . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied [13] . An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A (H1N1) pandemic revealed that the way plans were framed varied considerably, and '[told] us something about how the different countries want pandemics and preparedness to be understood by the public' [14] .", "An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A (H1N1) pandemic revealed that the way plans were framed varied considerably, and '[told] us something about how the different countries want pandemics and preparedness to be understood by the public' [14] . More research was encouraged into cultural and social structures in the respective countries. In Iceland, information about the Ebola epidemic in West Africa came from several sources.", "In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health (DH) first reported on the epidemic on 8 April 2014 [15] . In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered.", "In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered. Fear of a global epidemic was rife, and in media and online discussions, doubts were raised about the Icelandic health system´s capacity to take care of a patient with Ebola [16] [17] [18] , despite its ranking as one of the best in the world [16] . On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola [19] .", "On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola [19] . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital (LSH), the Department of Civil Protection and Emergency Management (DCPEM), and the seven Primary Healthcare Regional Organizations in the country at the time. Key external partners were the European Centre for Disease Prevention and Control (ECDC) and WHO, in addition to Nordic collaborators in epidemic preparedness [20] .", "Key external partners were the European Centre for Disease Prevention and Control (ECDC) and WHO, in addition to Nordic collaborators in epidemic preparedness [20] . At the same time, it was regarded as highly unlikely that Ebola Virus Disease (EVD) would spread in the country [21] . Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities [22] [23] [24] [25] .", "Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities [22] [23] [24] [25] . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa. Parked in a closed-off area, a physician in full Personal Protective Equipment (PPE) entered the plane, but quickly ruled out Ebola [26] .", "Parked in a closed-off area, a physician in full Personal Protective Equipment (PPE) entered the plane, but quickly ruled out Ebola [26] . Irrespective of good or bad overall performance, health systems are tested in times of crisis, such as epidemics. Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process.", "Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland [16, 27] . Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training.", "Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training. The study was conducted in April-May 2016, and the interviewed participants were administrators and frontline health professionals central to the case, so as to explore their perspectives and experiences concerning Ebola preparedness [28, 29] . Staff in managerial positions were contacted by one of the authors (GG) for permission to interview them based on their role in the preparedness plan.", "Staff in managerial positions were contacted by one of the authors (GG) for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team (ETT), the director of the team listed relevant email contacts. Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors (ÍEH), a physiotherapist.", "Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors (ÍEH), a physiotherapist. In case interviewees suggested other potential participants, they were invited through email to participate. A similar methodology was applied to identify participants from the Emergency Room (ER).", "A similar methodology was applied to identify participants from the Emergency Room (ER). They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness. Three separate interview guides were developedone each for managers, ETT, and ER respectively (see supplementary material).", "Three separate interview guides were developedone each for managers, ETT, and ER respectively (see supplementary material). The interviews included open questions probing the role of their institution in preparedness, the experience of the training process, challenges encountered or expected, and any dilemmas that they may have experienced in relation to the preparedness plan. The recruitment of participants was concluded when saturation was reached.", "The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis. The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views.", "The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views. From this exercise, three broad themes were inductively identified that corresponded to critical perspectives introduced by the participants. Permission to conduct the study was granted by Iceland's National Bioethics Committee (VSN- and Landspitali University Hospital (LSH 13-16, 4 February 2016) .", "Permission to conduct the study was granted by Iceland's National Bioethics Committee (VSN- and Landspitali University Hospital (LSH 13-16, 4 February 2016) . Reporting on the results was guided by the COREC guidelines [30] ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations. The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH [31] , and the preparatory activities engaged more than two hundred staff across all of its departments.", "The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH [31] , and the preparatory activities engaged more than two hundred staff across all of its departments. The ETT consisted of about 50 healthcare professionals who had volunteered to participate, including 11 doctors and 28 nurses, a few laboratory technicians, radiologists, and auxiliary nurses. They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care.", "They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD [31] . Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan.", "Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan. One manager concluded:\n\nYou get no discount. You can never go the shorter way. There was always something that surprised you.", "You can never go the shorter way. There was always something that surprised you. We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems.", "We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems. The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'.", "Ebola preparedness was 'a job that had to be done', and 'someone had to do it'. Some referred to ethical or professional obligations: This is just a part of being a nurse, to encounter situations that can be dangerous to you or someone else, but you have made this decision and you deal with it. Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV.", "Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV. Yet, the practice of volunteering in the preparation was questioned. One participant said:\n\nWe learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with.", "One participant said:\n\nWe learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with. ER staff indicated that for them working in the ER was enough of a risk to take, no reason to expose oneself even more by joining the ETT, and appreciated that others had volunteered. All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways.", "All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways. Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear.", "Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear. Those in managerial positions had listened and taken their opinions into consideration. One said:\n\nThey were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were.", "One said:\n\nThey were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were. Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan.", "Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan. Facilities for isolation and treatment of patients with Ebola were less than perfect:\n\nWe assessed how we could use the department … and change it in just a few hours into some kind of an isolation unit that we could possibly use. Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere.", "Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere. There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory.", "Respondents believed that the training and education of the members of the ETT and ER had been satisfactory. They felt that it had been proportionate to the risk, while some were concerned about the lack of staff. Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment.", "Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment. Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as [we] could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients?", "There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients? When asked if Ebola was a real threat to the country, participants usually referred to how easy it was to travel the globe: 'Yeah, why not, the world is getting smaller'. Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland.", "Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland. One respondent said:\n\nPeople were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts.", "Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness [would have been] needed in the PPE practices'. It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT.", "It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT. When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE.", "Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said:\n\nWhat bothered most of us was how uncomfortable the PPE was and I think that made people nervous: \"How will I manage working in this for hours?\" Another described the donning/doffing process like a 'complicated ballroom dance'.", "Another described the donning/doffing process like a 'complicated ballroom dance'. Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these [non-clinical] people belonged in the team, because this is a very clinical environment in addition to having to be in this costume [PPE] with the risk of becoming infected by mistake.", "One participant said: I didn't think these [non-clinical] people belonged in the team, because this is a very clinical environment in addition to having to be in this costume [PPE] with the risk of becoming infected by mistake. Those with non-clinical background were, however, aware of their limitations: I realized that I would not be the one in the front, I would not be managing patients directly. The importance ascribed to teamwork was evident in relation to fear.", "The importance ascribed to teamwork was evident in relation to fear. Participants described fear of working with people they had not worked with before:\n\nThe weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, [we] help each other.", "Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, [we] help each other. Then you don't want to be working with someone who didn't show up. There were a lot of doctors who just dropped in, dropped out, and then dropped in again.", "There were a lot of doctors who just dropped in, dropped out, and then dropped in again. I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland.", "Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland. A major precaution was planning to avoid contact with the family while working with Ebola patients. One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew (s)he was working with an Ebola patient.", "One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew (s)he was working with an Ebola patient. For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team.", "ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team. 'The normal insurance for LHS staff would not cover everything if we were to become sick or even lose our lives.' Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT.", "Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT. One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need.", "Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need. Characteristics associated with Iceland and the Icelandic people were referred to repeatedly by participants. The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations.", "The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations. 'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made.", "even though compromises had to be made. Comparing how Iceland handled its responsibilities differently from other countries of a larger size was often brought up, both with pride in Iceland as a strong independent nation, and with insecurities about its capacity in comparison to other countries. It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role.", "It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role. As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors.", "Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors. Notions of Icelanders as resilient in spite of shortcomings were common; referring to the experience of preparedness planning and training, one health staff said:\n\nIt was very much the Icelandic way, we'll manage, we'll work it out, and there was so much ingenuity. This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement:\n\nWould it have worked?", "This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement:\n\nWould it have worked? Yes, it would have worked. Would it have been optimal?", "Yes, it would have worked. Would it have been optimal? We cannot say, it would have been optimal; we can say, it would have been sufficient.", "We cannot say, it would have been optimal; we can say, it would have been sufficient. In contrast to this, there were concerns about whether Icelandic aid workers falling ill in Ebolaaffected countries should be transferred to Iceland or to hospitals in other Nordic countries with better isolation units. Some of the participants trusted that patients with EVD would not be transferred to Iceland.", "Some of the participants trusted that patients with EVD would not be transferred to Iceland. One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country.", "We don't know what would have happened if they would have transferred an Icelander into the country. We don't have good enough isolation unitsyou are not supposed to send patients to a hospital that is less than 100%. I thought there was assurance in that.", "I thought there was assurance in that. During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe [32] , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic [33] , in the EU region [34, 35] , and the US [36] .", "Evaluations have, for example, been conducted in 11 African countries close to the epidemic [33] , in the EU region [34, 35] , and the US [36] . Here we present data from a qualitative case study on the process, and experiences with establishing a preparedness plan for Ebola in Iceland in 2014. Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response.", "Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response. The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' (theme 1) refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation.", "'Getting the job done' (theme 1) refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation. Using the metaphors of 'hard core' and 'soft periphery', Langley and Denis [37] emphasize the importance of relatively 'peripheral' concerns and processes for planning and implementation of new interventions. The hard core represents the actual intervention or goal, e.g.", "The hard core represents the actual intervention or goal, e.g. implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core.", "The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core. If the soft periphery is neglected, it will cause multiple challenges in the implementation process, and the benefit of the hard core, the intervention itself, may not transpire as anticipated. Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders.", "Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders. In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan.", "The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan. Comprehensive consultations and the involvement of many associated institutions were mentioned. Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g.", "Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g. when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' [38] .", "During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' [38] . The West African Ebola epidemic was covered widely by media [39] , and the fear of Ebola reached every corner of the world, exemplified by travel bans from affected countries, and trade barriers [40] , in contrast to the ongoing epidemic in the Democratic Republic of Congo [41, 42] . In our second theme, trust, doubt, and fear of health workers were represented.", "In our second theme, trust, doubt, and fear of health workers were represented. Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned.", "The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned. In preparedness efforts in other countries, scarcity of resources in relation to manpower demand and problems with training and protocols involving PPE were common challenges [35] . Similar problems were encountered in Iceland.", "Similar problems were encountered in Iceland. Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere [43] . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal.", "Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal. The members of the ETT pointed out that they had never worked together as a team under circumstances that demanded strict adherence to infectious control procedures. This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation [44] .", "This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation [44] . Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics [35] . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada [45, 46] .", "This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada [45, 46] . Anthropological work on emergency preparedness emphasizes the connectedness between health professionals, technological devices, and knowledge as a prerequisite for successful preparedness. Wolf and Hall present preparedness efforts as a form of governance that involves human bodies (those of health professionals), clinical architectures (e.g.", "Wolf and Hall present preparedness efforts as a form of governance that involves human bodies (those of health professionals), clinical architectures (e.g. isolation wards), and technical artefacts (gloves, protective suits, disinfectants, etc.) [47] .", "[47] . During preparedness training and implementation, 'nursing bodies are transformed into instruments of preparedness', and become part of infrastructural arrangements. Health professionals are, here, both vulnerable and powerful tools in the management of contamination.", "Health professionals are, here, both vulnerable and powerful tools in the management of contamination. The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation.", "In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation. Repeated enactment of receiving and treating a patient with Ebola within experienced and trustful teams would probably enhance such embodiment, provided that there is justified trust in the involved technology. In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested.", "In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested. In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland [48] .", "Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland [48] . Notions of the intrinsic character of Iceland, and of being Icelandic, serve to underscore certain points and explain positive and negative experiences with the preparedness plan. Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere.", "Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere. In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' (things always have a way of working out in the end). The preparedness plan had to be completed, and adapted to Iceland's particular global situation.", "The preparedness plan had to be completed, and adapted to Iceland's particular global situation. In the 21st century, the world has faced new epidemic threats, such as SARS, and old scourges such as the plague have resurfaced [38] . One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 [35] .", "One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 [35] . Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 [49] .", "A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 [49] . During the elaboration of these plans, communication among the different levels of the healthcare system and supporting agencies, such as the DCPEM, had been clearly defined, and proved to be useful in the preparedness for Ebola. Further, as found important in preparedness activities for pandemic influenzae elsewhere [44] , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical.", "Further, as found important in preparedness activities for pandemic influenzae elsewhere [44] , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical. It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics [38] . Iceland was far away from the epicentre of the Ebola epidemic in West Africa.", "Iceland was far away from the epicentre of the Ebola epidemic in West Africa. Yet this case study shows that health professionals felt the strain of possibly having to treat one or more patients with EVD. Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD.", "Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD. Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process.", "References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process. These references negotiate the role and character of the nation of Iceland, and its role in a globalized world, as both a small and isolated nation on one hand, and a central and capable one, on the other. The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination.", "The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination. This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola (or other contagious global health threats) as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline.", "This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline. Geir Gunnlaugsson conceptualized the study, and took part in all necessary steps towards its completion, such as analysis and interpretation of data, and writing the manuscript for submission. Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version.", "Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version. Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version.", "Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version. Dr. Gunnlaugsson reports he was the Chief Medical Officer (CMO) for Iceland, Directorate of Health, in the period 2010-2014. Other authors report no conflict of interest.", "Other authors report no conflict of interest. The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland (number VSI- ). Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 (number LSH [13] [14] [15] [16] .", "Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 (number LSH [13] [14] [15] [16] . Participants signed an informed consent form before taking part in the study. Not applicable.", "Participants signed an informed consent form before taking part in the study. Not applicable. The manuscript builds on the work of Íris Eva Hauksdóttir towards a MSc in Global Health, Section of Global Health, Department of Public Health, Copenhagen University, Denmark." ]

[ "Background: The Ebola epidemic in West Africa caused global fear and stirred up worldwide preparedness activities in countries sharing borders with those affected, and in geographically far-away countries such as Iceland. Objective: To describe and analyse Ebola preparedness activities within the Icelandic healthcare system, and to explore the perspectives and experiences of managers and frontline health workers. Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team.", "Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team. Results: Contextual factors such as culture and demography influenced preparedness, and contributed to the positive state of mind of participants, and ingenuity in using available resources for preparedness. While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland.", "While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families. There were also concerns about the teamwork and lack of commitment by some during training.", "There were also concerns about the teamwork and lack of commitment by some during training. Being a ‘tiny’ nation was seen as both an asset and a weakness in the preparation process. Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans.", "Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so. For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support.", "For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support. Text: Global health; prevention and control; public policy; qualitative evaluation; emergency responders; communicable diseases; emerging; fear Background On 8 August 2014, the World Health Organization declared the Ebola epidemic in West Africa as a Public Health Emergency of International Concern (PHEIC) under the International Health Regulations (IHR) [1] . All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems [2] [3] [4] .", "All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems [2] [3] [4] . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola [5] , there was a growing fear of transmission beyond the region. In breach of WHO recommendations and guidelines [6] , flights were cancelled and cross-border movement curtailed [7] .", "In breach of WHO recommendations and guidelines [6] , flights were cancelled and cross-border movement curtailed [7] . The epidemic caused public concern outside West Africa [8] , as fear and racism found fertile ground [9] [10] [11] , and in an effort to stop the international spread of the disease, all states were advised to be prepared to detect, investigate, and manage Ebola cases [1] . Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' [12] .", "Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' [12] . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied [13] . An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A (H1N1) pandemic revealed that the way plans were framed varied considerably, and '[told] us something about how the different countries want pandemics and preparedness to be understood by the public' [14] .", "An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A (H1N1) pandemic revealed that the way plans were framed varied considerably, and '[told] us something about how the different countries want pandemics and preparedness to be understood by the public' [14] . More research was encouraged into cultural and social structures in the respective countries. In Iceland, information about the Ebola epidemic in West Africa came from several sources.", "In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health (DH) first reported on the epidemic on 8 April 2014 [15] . In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered.", "In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered. Fear of a global epidemic was rife, and in media and online discussions, doubts were raised about the Icelandic health system´s capacity to take care of a patient with Ebola [16] [17] [18] , despite its ranking as one of the best in the world [16] . On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola [19] .", "On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola [19] . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital (LSH), the Department of Civil Protection and Emergency Management (DCPEM), and the seven Primary Healthcare Regional Organizations in the country at the time. Key external partners were the European Centre for Disease Prevention and Control (ECDC) and WHO, in addition to Nordic collaborators in epidemic preparedness [20] .", "Key external partners were the European Centre for Disease Prevention and Control (ECDC) and WHO, in addition to Nordic collaborators in epidemic preparedness [20] . At the same time, it was regarded as highly unlikely that Ebola Virus Disease (EVD) would spread in the country [21] . Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities [22] [23] [24] [25] .", "Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities [22] [23] [24] [25] . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa. Parked in a closed-off area, a physician in full Personal Protective Equipment (PPE) entered the plane, but quickly ruled out Ebola [26] .", "Parked in a closed-off area, a physician in full Personal Protective Equipment (PPE) entered the plane, but quickly ruled out Ebola [26] . Irrespective of good or bad overall performance, health systems are tested in times of crisis, such as epidemics. Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process.", "Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland [16, 27] . Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training.", "Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training. The study was conducted in April-May 2016, and the interviewed participants were administrators and frontline health professionals central to the case, so as to explore their perspectives and experiences concerning Ebola preparedness [28, 29] . Staff in managerial positions were contacted by one of the authors (GG) for permission to interview them based on their role in the preparedness plan.", "Staff in managerial positions were contacted by one of the authors (GG) for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team (ETT), the director of the team listed relevant email contacts. Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors (ÍEH), a physiotherapist.", "Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors (ÍEH), a physiotherapist. In case interviewees suggested other potential participants, they were invited through email to participate. A similar methodology was applied to identify participants from the Emergency Room (ER).", "A similar methodology was applied to identify participants from the Emergency Room (ER). They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness. Three separate interview guides were developedone each for managers, ETT, and ER respectively (see supplementary material).", "Three separate interview guides were developedone each for managers, ETT, and ER respectively (see supplementary material). The interviews included open questions probing the role of their institution in preparedness, the experience of the training process, challenges encountered or expected, and any dilemmas that they may have experienced in relation to the preparedness plan. The recruitment of participants was concluded when saturation was reached.", "The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis. The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views.", "The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views. From this exercise, three broad themes were inductively identified that corresponded to critical perspectives introduced by the participants. Permission to conduct the study was granted by Iceland's National Bioethics Committee (VSN- and Landspitali University Hospital (LSH 13-16, 4 February 2016) .", "Permission to conduct the study was granted by Iceland's National Bioethics Committee (VSN- and Landspitali University Hospital (LSH 13-16, 4 February 2016) . Reporting on the results was guided by the COREC guidelines [30] ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations. The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH [31] , and the preparatory activities engaged more than two hundred staff across all of its departments.", "The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH [31] , and the preparatory activities engaged more than two hundred staff across all of its departments. The ETT consisted of about 50 healthcare professionals who had volunteered to participate, including 11 doctors and 28 nurses, a few laboratory technicians, radiologists, and auxiliary nurses. They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care.", "They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD [31] . Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan.", "Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan. One manager concluded:\n\nYou get no discount. You can never go the shorter way. There was always something that surprised you.", "You can never go the shorter way. There was always something that surprised you. We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems.", "We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems. The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'.", "Ebola preparedness was 'a job that had to be done', and 'someone had to do it'. Some referred to ethical or professional obligations: This is just a part of being a nurse, to encounter situations that can be dangerous to you or someone else, but you have made this decision and you deal with it. Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV.", "Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV. Yet, the practice of volunteering in the preparation was questioned. One participant said:\n\nWe learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with.", "One participant said:\n\nWe learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with. ER staff indicated that for them working in the ER was enough of a risk to take, no reason to expose oneself even more by joining the ETT, and appreciated that others had volunteered. All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways.", "All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways. Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear.", "Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear. Those in managerial positions had listened and taken their opinions into consideration. One said:\n\nThey were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were.", "One said:\n\nThey were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were. Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan.", "Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan. Facilities for isolation and treatment of patients with Ebola were less than perfect:\n\nWe assessed how we could use the department … and change it in just a few hours into some kind of an isolation unit that we could possibly use. Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere.", "Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere. There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory.", "Respondents believed that the training and education of the members of the ETT and ER had been satisfactory. They felt that it had been proportionate to the risk, while some were concerned about the lack of staff. Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment.", "Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment. Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as [we] could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients?", "There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients? When asked if Ebola was a real threat to the country, participants usually referred to how easy it was to travel the globe: 'Yeah, why not, the world is getting smaller'. Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland.", "Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland. One respondent said:\n\nPeople were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts.", "Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness [would have been] needed in the PPE practices'. It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT.", "It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT. When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE.", "Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said:\n\nWhat bothered most of us was how uncomfortable the PPE was and I think that made people nervous: \"How will I manage working in this for hours?\" Another described the donning/doffing process like a 'complicated ballroom dance'.", "Another described the donning/doffing process like a 'complicated ballroom dance'. Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these [non-clinical] people belonged in the team, because this is a very clinical environment in addition to having to be in this costume [PPE] with the risk of becoming infected by mistake.", "One participant said: I didn't think these [non-clinical] people belonged in the team, because this is a very clinical environment in addition to having to be in this costume [PPE] with the risk of becoming infected by mistake. Those with non-clinical background were, however, aware of their limitations: I realized that I would not be the one in the front, I would not be managing patients directly. The importance ascribed to teamwork was evident in relation to fear.", "The importance ascribed to teamwork was evident in relation to fear. Participants described fear of working with people they had not worked with before:\n\nThe weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, [we] help each other.", "Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, [we] help each other. Then you don't want to be working with someone who didn't show up. There were a lot of doctors who just dropped in, dropped out, and then dropped in again.", "There were a lot of doctors who just dropped in, dropped out, and then dropped in again. I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland.", "Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland. A major precaution was planning to avoid contact with the family while working with Ebola patients. One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew (s)he was working with an Ebola patient.", "One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew (s)he was working with an Ebola patient. For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team.", "ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team. 'The normal insurance for LHS staff would not cover everything if we were to become sick or even lose our lives.' Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT.", "Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT. One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need.", "Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need. Characteristics associated with Iceland and the Icelandic people were referred to repeatedly by participants. The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations.", "The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations. 'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made.", "even though compromises had to be made. Comparing how Iceland handled its responsibilities differently from other countries of a larger size was often brought up, both with pride in Iceland as a strong independent nation, and with insecurities about its capacity in comparison to other countries. It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role.", "It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role. As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors.", "Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors. Notions of Icelanders as resilient in spite of shortcomings were common; referring to the experience of preparedness planning and training, one health staff said:\n\nIt was very much the Icelandic way, we'll manage, we'll work it out, and there was so much ingenuity. This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement:\n\nWould it have worked?", "This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement:\n\nWould it have worked? Yes, it would have worked. Would it have been optimal?", "Yes, it would have worked. Would it have been optimal? We cannot say, it would have been optimal; we can say, it would have been sufficient.", "We cannot say, it would have been optimal; we can say, it would have been sufficient. In contrast to this, there were concerns about whether Icelandic aid workers falling ill in Ebolaaffected countries should be transferred to Iceland or to hospitals in other Nordic countries with better isolation units. Some of the participants trusted that patients with EVD would not be transferred to Iceland.", "Some of the participants trusted that patients with EVD would not be transferred to Iceland. One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country.", "We don't know what would have happened if they would have transferred an Icelander into the country. We don't have good enough isolation unitsyou are not supposed to send patients to a hospital that is less than 100%. I thought there was assurance in that.", "I thought there was assurance in that. During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe [32] , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic [33] , in the EU region [34, 35] , and the US [36] .", "Evaluations have, for example, been conducted in 11 African countries close to the epidemic [33] , in the EU region [34, 35] , and the US [36] . Here we present data from a qualitative case study on the process, and experiences with establishing a preparedness plan for Ebola in Iceland in 2014. Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response.", "Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response. The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' (theme 1) refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation.", "'Getting the job done' (theme 1) refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation. Using the metaphors of 'hard core' and 'soft periphery', Langley and Denis [37] emphasize the importance of relatively 'peripheral' concerns and processes for planning and implementation of new interventions. The hard core represents the actual intervention or goal, e.g.", "The hard core represents the actual intervention or goal, e.g. implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core.", "The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core. If the soft periphery is neglected, it will cause multiple challenges in the implementation process, and the benefit of the hard core, the intervention itself, may not transpire as anticipated. Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders.", "Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders. In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan.", "The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan. Comprehensive consultations and the involvement of many associated institutions were mentioned. Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g.", "Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g. when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' [38] .", "During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' [38] . The West African Ebola epidemic was covered widely by media [39] , and the fear of Ebola reached every corner of the world, exemplified by travel bans from affected countries, and trade barriers [40] , in contrast to the ongoing epidemic in the Democratic Republic of Congo [41, 42] . In our second theme, trust, doubt, and fear of health workers were represented.", "In our second theme, trust, doubt, and fear of health workers were represented. Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned.", "The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned. In preparedness efforts in other countries, scarcity of resources in relation to manpower demand and problems with training and protocols involving PPE were common challenges [35] . Similar problems were encountered in Iceland.", "Similar problems were encountered in Iceland. Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere [43] . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal.", "Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal. The members of the ETT pointed out that they had never worked together as a team under circumstances that demanded strict adherence to infectious control procedures. This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation [44] .", "This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation [44] . Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics [35] . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada [45, 46] .", "This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada [45, 46] . Anthropological work on emergency preparedness emphasizes the connectedness between health professionals, technological devices, and knowledge as a prerequisite for successful preparedness. Wolf and Hall present preparedness efforts as a form of governance that involves human bodies (those of health professionals), clinical architectures (e.g.", "Wolf and Hall present preparedness efforts as a form of governance that involves human bodies (those of health professionals), clinical architectures (e.g. isolation wards), and technical artefacts (gloves, protective suits, disinfectants, etc.) [47] .", "[47] . During preparedness training and implementation, 'nursing bodies are transformed into instruments of preparedness', and become part of infrastructural arrangements. Health professionals are, here, both vulnerable and powerful tools in the management of contamination.", "Health professionals are, here, both vulnerable and powerful tools in the management of contamination. The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation.", "In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation. Repeated enactment of receiving and treating a patient with Ebola within experienced and trustful teams would probably enhance such embodiment, provided that there is justified trust in the involved technology. In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested.", "In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested. In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland [48] .", "Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland [48] . Notions of the intrinsic character of Iceland, and of being Icelandic, serve to underscore certain points and explain positive and negative experiences with the preparedness plan. Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere.", "Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere. In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' (things always have a way of working out in the end). The preparedness plan had to be completed, and adapted to Iceland's particular global situation.", "The preparedness plan had to be completed, and adapted to Iceland's particular global situation. In the 21st century, the world has faced new epidemic threats, such as SARS, and old scourges such as the plague have resurfaced [38] . One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 [35] .", "One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 [35] . Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 [49] .", "A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 [49] . During the elaboration of these plans, communication among the different levels of the healthcare system and supporting agencies, such as the DCPEM, had been clearly defined, and proved to be useful in the preparedness for Ebola. Further, as found important in preparedness activities for pandemic influenzae elsewhere [44] , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical.", "Further, as found important in preparedness activities for pandemic influenzae elsewhere [44] , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical. It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics [38] . Iceland was far away from the epicentre of the Ebola epidemic in West Africa.", "Iceland was far away from the epicentre of the Ebola epidemic in West Africa. Yet this case study shows that health professionals felt the strain of possibly having to treat one or more patients with EVD. Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD.", "Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD. Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process.", "References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process. These references negotiate the role and character of the nation of Iceland, and its role in a globalized world, as both a small and isolated nation on one hand, and a central and capable one, on the other. The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination.", "The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination. This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola (or other contagious global health threats) as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline.", "This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline. Geir Gunnlaugsson conceptualized the study, and took part in all necessary steps towards its completion, such as analysis and interpretation of data, and writing the manuscript for submission. Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version.", "Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version. Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version.", "Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version. Dr. Gunnlaugsson reports he was the Chief Medical Officer (CMO) for Iceland, Directorate of Health, in the period 2010-2014. Other authors report no conflict of interest.", "Other authors report no conflict of interest. The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland (number VSI- ). Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 (number LSH [13] [14] [15] [16] .", "Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 (number LSH [13] [14] [15] [16] . Participants signed an informed consent form before taking part in the study. Not applicable.", "Participants signed an informed consent form before taking part in the study. Not applicable. The manuscript builds on the work of Íris Eva Hauksdóttir towards a MSc in Global Health, Section of Global Health, Department of Public Health, Copenhagen University, Denmark." ]

[ "Background: The Ebola epidemic in West Africa caused global fear and stirred up worldwide preparedness activities in countries sharing borders with those affected, and in geographically far-away countries such as Iceland. Objective: To describe and analyse Ebola preparedness activities within the Icelandic healthcare system, and to explore the perspectives and experiences of managers and frontline health workers. Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team.", "Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team. Results: Contextual factors such as culture and demography influenced preparedness, and contributed to the positive state of mind of participants, and ingenuity in using available resources for preparedness. While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland.", "While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families. There were also concerns about the teamwork and lack of commitment by some during training.", "There were also concerns about the teamwork and lack of commitment by some during training. Being a ‘tiny’ nation was seen as both an asset and a weakness in the preparation process. Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans.", "Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so. For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support.", "For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support. Text: Global health; prevention and control; public policy; qualitative evaluation; emergency responders; communicable diseases; emerging; fear Background On 8 August 2014, the World Health Organization declared the Ebola epidemic in West Africa as a Public Health Emergency of International Concern (PHEIC) under the International Health Regulations (IHR) [1] . All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems [2] [3] [4] .", "All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems [2] [3] [4] . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola [5] , there was a growing fear of transmission beyond the region. In breach of WHO recommendations and guidelines [6] , flights were cancelled and cross-border movement curtailed [7] .", "In breach of WHO recommendations and guidelines [6] , flights were cancelled and cross-border movement curtailed [7] . The epidemic caused public concern outside West Africa [8] , as fear and racism found fertile ground [9] [10] [11] , and in an effort to stop the international spread of the disease, all states were advised to be prepared to detect, investigate, and manage Ebola cases [1] . Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' [12] .", "Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' [12] . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied [13] . An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A (H1N1) pandemic revealed that the way plans were framed varied considerably, and '[told] us something about how the different countries want pandemics and preparedness to be understood by the public' [14] .", "An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A (H1N1) pandemic revealed that the way plans were framed varied considerably, and '[told] us something about how the different countries want pandemics and preparedness to be understood by the public' [14] . More research was encouraged into cultural and social structures in the respective countries. In Iceland, information about the Ebola epidemic in West Africa came from several sources.", "In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health (DH) first reported on the epidemic on 8 April 2014 [15] . In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered.", "In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered. Fear of a global epidemic was rife, and in media and online discussions, doubts were raised about the Icelandic health system´s capacity to take care of a patient with Ebola [16] [17] [18] , despite its ranking as one of the best in the world [16] . On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola [19] .", "On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola [19] . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital (LSH), the Department of Civil Protection and Emergency Management (DCPEM), and the seven Primary Healthcare Regional Organizations in the country at the time. Key external partners were the European Centre for Disease Prevention and Control (ECDC) and WHO, in addition to Nordic collaborators in epidemic preparedness [20] .", "Key external partners were the European Centre for Disease Prevention and Control (ECDC) and WHO, in addition to Nordic collaborators in epidemic preparedness [20] . At the same time, it was regarded as highly unlikely that Ebola Virus Disease (EVD) would spread in the country [21] . Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities [22] [23] [24] [25] .", "Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities [22] [23] [24] [25] . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa. Parked in a closed-off area, a physician in full Personal Protective Equipment (PPE) entered the plane, but quickly ruled out Ebola [26] .", "Parked in a closed-off area, a physician in full Personal Protective Equipment (PPE) entered the plane, but quickly ruled out Ebola [26] . Irrespective of good or bad overall performance, health systems are tested in times of crisis, such as epidemics. Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process.", "Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland [16, 27] . Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training.", "Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training. The study was conducted in April-May 2016, and the interviewed participants were administrators and frontline health professionals central to the case, so as to explore their perspectives and experiences concerning Ebola preparedness [28, 29] . Staff in managerial positions were contacted by one of the authors (GG) for permission to interview them based on their role in the preparedness plan.", "Staff in managerial positions were contacted by one of the authors (GG) for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team (ETT), the director of the team listed relevant email contacts. Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors (ÍEH), a physiotherapist.", "Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors (ÍEH), a physiotherapist. In case interviewees suggested other potential participants, they were invited through email to participate. A similar methodology was applied to identify participants from the Emergency Room (ER).", "A similar methodology was applied to identify participants from the Emergency Room (ER). They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness. Three separate interview guides were developedone each for managers, ETT, and ER respectively (see supplementary material).", "Three separate interview guides were developedone each for managers, ETT, and ER respectively (see supplementary material). The interviews included open questions probing the role of their institution in preparedness, the experience of the training process, challenges encountered or expected, and any dilemmas that they may have experienced in relation to the preparedness plan. The recruitment of participants was concluded when saturation was reached.", "The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis. The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views.", "The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views. From this exercise, three broad themes were inductively identified that corresponded to critical perspectives introduced by the participants. Permission to conduct the study was granted by Iceland's National Bioethics Committee (VSN- and Landspitali University Hospital (LSH 13-16, 4 February 2016) .", "Permission to conduct the study was granted by Iceland's National Bioethics Committee (VSN- and Landspitali University Hospital (LSH 13-16, 4 February 2016) . Reporting on the results was guided by the COREC guidelines [30] ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations. The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH [31] , and the preparatory activities engaged more than two hundred staff across all of its departments.", "The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH [31] , and the preparatory activities engaged more than two hundred staff across all of its departments. The ETT consisted of about 50 healthcare professionals who had volunteered to participate, including 11 doctors and 28 nurses, a few laboratory technicians, radiologists, and auxiliary nurses. They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care.", "They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD [31] . Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan.", "Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan. One manager concluded:\n\nYou get no discount. You can never go the shorter way. There was always something that surprised you.", "You can never go the shorter way. There was always something that surprised you. We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems.", "We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems. The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'.", "Ebola preparedness was 'a job that had to be done', and 'someone had to do it'. Some referred to ethical or professional obligations: This is just a part of being a nurse, to encounter situations that can be dangerous to you or someone else, but you have made this decision and you deal with it. Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV.", "Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV. Yet, the practice of volunteering in the preparation was questioned. One participant said:\n\nWe learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with.", "One participant said:\n\nWe learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with. ER staff indicated that for them working in the ER was enough of a risk to take, no reason to expose oneself even more by joining the ETT, and appreciated that others had volunteered. All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways.", "All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways. Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear.", "Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear. Those in managerial positions had listened and taken their opinions into consideration. One said:\n\nThey were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were.", "One said:\n\nThey were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were. Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan.", "Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan. Facilities for isolation and treatment of patients with Ebola were less than perfect:\n\nWe assessed how we could use the department … and change it in just a few hours into some kind of an isolation unit that we could possibly use. Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere.", "Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere. There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory.", "Respondents believed that the training and education of the members of the ETT and ER had been satisfactory. They felt that it had been proportionate to the risk, while some were concerned about the lack of staff. Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment.", "Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment. Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as [we] could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients?", "There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients? When asked if Ebola was a real threat to the country, participants usually referred to how easy it was to travel the globe: 'Yeah, why not, the world is getting smaller'. Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland.", "Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland. One respondent said:\n\nPeople were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts.", "Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness [would have been] needed in the PPE practices'. It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT.", "It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT. When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE.", "Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said:\n\nWhat bothered most of us was how uncomfortable the PPE was and I think that made people nervous: \"How will I manage working in this for hours?\" Another described the donning/doffing process like a 'complicated ballroom dance'.", "Another described the donning/doffing process like a 'complicated ballroom dance'. Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these [non-clinical] people belonged in the team, because this is a very clinical environment in addition to having to be in this costume [PPE] with the risk of becoming infected by mistake.", "One participant said: I didn't think these [non-clinical] people belonged in the team, because this is a very clinical environment in addition to having to be in this costume [PPE] with the risk of becoming infected by mistake. Those with non-clinical background were, however, aware of their limitations: I realized that I would not be the one in the front, I would not be managing patients directly. The importance ascribed to teamwork was evident in relation to fear.", "The importance ascribed to teamwork was evident in relation to fear. Participants described fear of working with people they had not worked with before:\n\nThe weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, [we] help each other.", "Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, [we] help each other. Then you don't want to be working with someone who didn't show up. There were a lot of doctors who just dropped in, dropped out, and then dropped in again.", "There were a lot of doctors who just dropped in, dropped out, and then dropped in again. I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland.", "Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland. A major precaution was planning to avoid contact with the family while working with Ebola patients. One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew (s)he was working with an Ebola patient.", "One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew (s)he was working with an Ebola patient. For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team.", "ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team. 'The normal insurance for LHS staff would not cover everything if we were to become sick or even lose our lives.' Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT.", "Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT. One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need.", "Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need. Characteristics associated with Iceland and the Icelandic people were referred to repeatedly by participants. The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations.", "The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations. 'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made.", "even though compromises had to be made. Comparing how Iceland handled its responsibilities differently from other countries of a larger size was often brought up, both with pride in Iceland as a strong independent nation, and with insecurities about its capacity in comparison to other countries. It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role.", "It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role. As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors.", "Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors. Notions of Icelanders as resilient in spite of shortcomings were common; referring to the experience of preparedness planning and training, one health staff said:\n\nIt was very much the Icelandic way, we'll manage, we'll work it out, and there was so much ingenuity. This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement:\n\nWould it have worked?", "This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement:\n\nWould it have worked? Yes, it would have worked. Would it have been optimal?", "Yes, it would have worked. Would it have been optimal? We cannot say, it would have been optimal; we can say, it would have been sufficient.", "We cannot say, it would have been optimal; we can say, it would have been sufficient. In contrast to this, there were concerns about whether Icelandic aid workers falling ill in Ebolaaffected countries should be transferred to Iceland or to hospitals in other Nordic countries with better isolation units. Some of the participants trusted that patients with EVD would not be transferred to Iceland.", "Some of the participants trusted that patients with EVD would not be transferred to Iceland. One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country.", "We don't know what would have happened if they would have transferred an Icelander into the country. We don't have good enough isolation unitsyou are not supposed to send patients to a hospital that is less than 100%. I thought there was assurance in that.", "I thought there was assurance in that. During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe [32] , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic [33] , in the EU region [34, 35] , and the US [36] .", "Evaluations have, for example, been conducted in 11 African countries close to the epidemic [33] , in the EU region [34, 35] , and the US [36] . Here we present data from a qualitative case study on the process, and experiences with establishing a preparedness plan for Ebola in Iceland in 2014. Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response.", "Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response. The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' (theme 1) refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation.", "'Getting the job done' (theme 1) refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation. Using the metaphors of 'hard core' and 'soft periphery', Langley and Denis [37] emphasize the importance of relatively 'peripheral' concerns and processes for planning and implementation of new interventions. The hard core represents the actual intervention or goal, e.g.", "The hard core represents the actual intervention or goal, e.g. implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core.", "The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core. If the soft periphery is neglected, it will cause multiple challenges in the implementation process, and the benefit of the hard core, the intervention itself, may not transpire as anticipated. Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders.", "Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders. In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan.", "The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan. Comprehensive consultations and the involvement of many associated institutions were mentioned. Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g.", "Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g. when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' [38] .", "During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' [38] . The West African Ebola epidemic was covered widely by media [39] , and the fear of Ebola reached every corner of the world, exemplified by travel bans from affected countries, and trade barriers [40] , in contrast to the ongoing epidemic in the Democratic Republic of Congo [41, 42] . In our second theme, trust, doubt, and fear of health workers were represented.", "In our second theme, trust, doubt, and fear of health workers were represented. Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned.", "The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned. In preparedness efforts in other countries, scarcity of resources in relation to manpower demand and problems with training and protocols involving PPE were common challenges [35] . Similar problems were encountered in Iceland.", "Similar problems were encountered in Iceland. Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere [43] . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal.", "Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal. The members of the ETT pointed out that they had never worked together as a team under circumstances that demanded strict adherence to infectious control procedures. This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation [44] .", "This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation [44] . Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics [35] . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada [45, 46] .", "This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada [45, 46] . Anthropological work on emergency preparedness emphasizes the connectedness between health professionals, technological devices, and knowledge as a prerequisite for successful preparedness. Wolf and Hall present preparedness efforts as a form of governance that involves human bodies (those of health professionals), clinical architectures (e.g.", "Wolf and Hall present preparedness efforts as a form of governance that involves human bodies (those of health professionals), clinical architectures (e.g. isolation wards), and technical artefacts (gloves, protective suits, disinfectants, etc.) [47] .", "[47] . During preparedness training and implementation, 'nursing bodies are transformed into instruments of preparedness', and become part of infrastructural arrangements. Health professionals are, here, both vulnerable and powerful tools in the management of contamination.", "Health professionals are, here, both vulnerable and powerful tools in the management of contamination. The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation.", "In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation. Repeated enactment of receiving and treating a patient with Ebola within experienced and trustful teams would probably enhance such embodiment, provided that there is justified trust in the involved technology. In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested.", "In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested. In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland [48] .", "Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland [48] . Notions of the intrinsic character of Iceland, and of being Icelandic, serve to underscore certain points and explain positive and negative experiences with the preparedness plan. Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere.", "Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere. In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' (things always have a way of working out in the end). The preparedness plan had to be completed, and adapted to Iceland's particular global situation.", "The preparedness plan had to be completed, and adapted to Iceland's particular global situation. In the 21st century, the world has faced new epidemic threats, such as SARS, and old scourges such as the plague have resurfaced [38] . One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 [35] .", "One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 [35] . Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 [49] .", "A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 [49] . During the elaboration of these plans, communication among the different levels of the healthcare system and supporting agencies, such as the DCPEM, had been clearly defined, and proved to be useful in the preparedness for Ebola. Further, as found important in preparedness activities for pandemic influenzae elsewhere [44] , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical.", "Further, as found important in preparedness activities for pandemic influenzae elsewhere [44] , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical. It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics [38] . Iceland was far away from the epicentre of the Ebola epidemic in West Africa.", "Iceland was far away from the epicentre of the Ebola epidemic in West Africa. Yet this case study shows that health professionals felt the strain of possibly having to treat one or more patients with EVD. Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD.", "Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD. Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process.", "References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process. These references negotiate the role and character of the nation of Iceland, and its role in a globalized world, as both a small and isolated nation on one hand, and a central and capable one, on the other. The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination.", "The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination. This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola (or other contagious global health threats) as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline.", "This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline. Geir Gunnlaugsson conceptualized the study, and took part in all necessary steps towards its completion, such as analysis and interpretation of data, and writing the manuscript for submission. Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version.", "Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version. Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version.", "Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version. Dr. Gunnlaugsson reports he was the Chief Medical Officer (CMO) for Iceland, Directorate of Health, in the period 2010-2014. Other authors report no conflict of interest.", "Other authors report no conflict of interest. The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland (number VSI- ). Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 (number LSH [13] [14] [15] [16] .", "Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 (number LSH [13] [14] [15] [16] . Participants signed an informed consent form before taking part in the study. Not applicable.", "Participants signed an informed consent form before taking part in the study. Not applicable. The manuscript builds on the work of Íris Eva Hauksdóttir towards a MSc in Global Health, Section of Global Health, Department of Public Health, Copenhagen University, Denmark." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus.", "This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58.", "Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] .", "Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] .", "The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected.", "Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] .", "There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models.", "However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2.", "Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al.", "As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day.", "The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far.", "While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 .", "SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model\n\nThe BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals).", "We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) .", "1) . The BHRP transmission network model was based on the following assumptions or facts:\n\na) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B .", "The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B .", "The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ).", "b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts.", "In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively.", "The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability.", "We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.", "d) The people were divided into five compartments:\n\nsusceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people.", "In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P .", "The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively.", "The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 .", "The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored.", "Therefore, we added the further assumptions as follows:\n\na) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function:\n\nIn the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level.", "Therefore, the BHRP model was simplified as RP model and is shown as follows:\n\nDuring the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively.", "Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P .", "Based on our previous research [15] , we therefore used the following sets to perform the normalization:\n\nIn the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows:\n\nThe transmissibility of the SARS-CoV-2 based on the RP model\n\nIn this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] .", "Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end.", "If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P .", "The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions:\n\na) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study.", "We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] .", "The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5).", "Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5.", "We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] .", "As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020.", "We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model.", "Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10.", "However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data.", "f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market.", "h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P .", "In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix:\n\nBy the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: \n\nThe R 0 of the normalized RP model is shown as follows:\n\nOur modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) .", "2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively.", "In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person.", "The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] .", "Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] .", "employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person.", "Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission.", "However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] .", "Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS.", "The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] .", "However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 .", "To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ.", "The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission.", "All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] .", "Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%.", "It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000.", "Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct.", "In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus.", "Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature.", "Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately." ]

[ "In early December 2019 a cluster of cases of pneumonia of unknown cause was identified in Wuhan, a city of 11 million persons in the People&rsquo;s Republic of China. Further investigation revealed these cases to result from infection with a newly identified coronavirus, termed the 2019-nCoV. The infection moved rapidly through China, spread to Thailand and Japan, extended into adjacent countries through infected persons travelling by air, eventually reaching multiple countries and continents.", "The infection moved rapidly through China, spread to Thailand and Japan, extended into adjacent countries through infected persons travelling by air, eventually reaching multiple countries and continents. Similar to such other coronaviruses as those causing the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS), the new coronavirus was reported to spread via natural aerosols from human-to-human. In the early stages of this epidemic the case fatality rate is estimated to be approximately 2%, with the majority of deaths occurring in special populations.", "In the early stages of this epidemic the case fatality rate is estimated to be approximately 2%, with the majority of deaths occurring in special populations. Unfortunately, there is limited experience with coronavirus infections during pregnancy, and it now appears certain that pregnant women have become infected during the present 2019-nCoV epidemic. In order to assess the potential of the Wuhan 2019-nCoV to cause maternal, fetal and neonatal morbidity and other poor obstetrical outcomes, this communication reviews the published data addressing the epidemiological and clinical effects of SARS, MERS, and other coronavirus infections on pregnant women and their infants.", "In order to assess the potential of the Wuhan 2019-nCoV to cause maternal, fetal and neonatal morbidity and other poor obstetrical outcomes, this communication reviews the published data addressing the epidemiological and clinical effects of SARS, MERS, and other coronavirus infections on pregnant women and their infants. Recommendations are also made for the consideration of pregnant women in the design, clinical trials, and implementation of future 2019-nCoV vaccines. Text: Coronaviruses are spherical, enveloped, and the largest of positive-strand RNA viruses.", "Text: Coronaviruses are spherical, enveloped, and the largest of positive-strand RNA viruses. They have a wide host range, including birds, farm animals, pets, camels, and bats, in which they primarily cause respiratory and gastrointestinal disease. Belonging to the order Nidovirales, family Coronaviridae, and the subfamily Orthocoronaviridae there are four genera of coronaviruses-Alphacoronavirus, Betacoronavirus, Deltacorona virus, and Gammacoronavirus [1] [2] [3] [4] .", "Belonging to the order Nidovirales, family Coronaviridae, and the subfamily Orthocoronaviridae there are four genera of coronaviruses-Alphacoronavirus, Betacoronavirus, Deltacorona virus, and Gammacoronavirus [1] [2] [3] [4] . In humans, they are a cause of mild illnesses including the common colds occurring in children and adults, and were believed to be of modest medical importance. However, two zoonotic coronaviruses-including the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV)-can produce severe lower respiratory In the beginning of December 2019, a cluster of persons with a pneumonia of unknown cause was identified in Wuhan, the capital of Hubei Province and a large city of approximately 11 million persons located in the central region of the People's Republic of China [7, 8] .", "However, two zoonotic coronaviruses-including the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV)-can produce severe lower respiratory In the beginning of December 2019, a cluster of persons with a pneumonia of unknown cause was identified in Wuhan, the capital of Hubei Province and a large city of approximately 11 million persons located in the central region of the People's Republic of China [7, 8] . Between 8 and 18 December 2019 there were 7 cases of pneumonia identified whose clinical features resembled that of a viral pneumonia. The outbreak was initially believed to be linked to the Wuhan Huanan (South China) Seafood Wholesale Market.", "The outbreak was initially believed to be linked to the Wuhan Huanan (South China) Seafood Wholesale Market. This market, termed a \"wet\" market, sells a variety of seafood, cuts of meat, and both live and dead animals in over one thousand stalls in constant close contact; however, whether this market was the origin of the outbreak remains unknown [9] . On 31 December 2019, the Chinese Center for Disease Control and Prevention (China CDC) sent a rapid response team to Hubei to work alongside health personnel from the provincial and Wuhan city health departments to conduct an epidemiologic investigation.", "On 31 December 2019, the Chinese Center for Disease Control and Prevention (China CDC) sent a rapid response team to Hubei to work alongside health personnel from the provincial and Wuhan city health departments to conduct an epidemiologic investigation. As the disease was spreading through secondary and tertiary cases, the World Health Organization (WHO) China Country Office was informed on 31 December 2019 of the occurrence of these cases of pneumonia of unknown etiology. During the period from 31 December 2019 to 3 January 2020, 44 patients with pneumonia of unknown etiology were reported by the Chinese authorities to the WHO.", "During the period from 31 December 2019 to 3 January 2020, 44 patients with pneumonia of unknown etiology were reported by the Chinese authorities to the WHO. On 7 January 2020 investigators in China identified the etiological agent of the epidemic as a previously unknown coronavirus, and it was given the designation 2019-nCoV (for 2019 novel coronavirus) [8] . Analysis of the clinical features of 41 hospitalized patients with laboratory-confirmed 2019-nCoV infection revealed that 30 were men (73%); less than one-half had underlying co-morbid conditions (13; 32%) which included diabetes (8, 20%) , hypertension (6, 15%), and cardiovascular disease (6; 15%); and the average age was 49.0 years old.", "Analysis of the clinical features of 41 hospitalized patients with laboratory-confirmed 2019-nCoV infection revealed that 30 were men (73%); less than one-half had underlying co-morbid conditions (13; 32%) which included diabetes (8, 20%) , hypertension (6, 15%), and cardiovascular disease (6; 15%); and the average age was 49.0 years old. The most common symptoms at the beginning of their illness included fever (40, 98%) , cough (31, 76%) , and fatigue or myalgia (18, 44%) , sputum production (11, 28%) , and headache (3, 8%) [10] . Among these 41 initial cases of 2019-nCoV infection there were 12 patients (32%) who developed acute respiratory distress syndrome (ARDS), 13 (32%) required intensive care and 6 (15%) died.", "Among these 41 initial cases of 2019-nCoV infection there were 12 patients (32%) who developed acute respiratory distress syndrome (ARDS), 13 (32%) required intensive care and 6 (15%) died. During the first weeks of January the infection spread rapidly through China and extended to adjacent countries where cases began to appear-13 January in Thailand, 15 January in Japan, 20 January in the Republic of Korea, and Taiwan and the United States on 21 January [11] . Infected travelers, mostly via commercial air travel, are known to have been responsible for introducing the virus outside of Wuhan.", "Infected travelers, mostly via commercial air travel, are known to have been responsible for introducing the virus outside of Wuhan. The new coronavirus continued to spread throughout multiple countries and continents, and by 9 February 2020 the WHO reported 37,251 confirmed cases in China that resulted in 812 deaths, surpassing the number of deaths that occurred during the 2002-2003 SARS epidemic. An additional 307 cases of 2019-nCoV infection have occurred among 24 other countries outside of China [12] .", "An additional 307 cases of 2019-nCoV infection have occurred among 24 other countries outside of China [12] . (Figure 1 ) At the meeting of the Emergency Committee of the WHO on 30 January, the novel coronavirus 2019 epidemic was declared a Public Health Emergency of International Concern (PHEIC) [11, 13] . Viruses 2020, 12, 194 3 of 16 epidemic.", "Viruses 2020, 12, 194 3 of 16 epidemic. An additional 307 cases of 2019-nCoV infection have occurred among 24 other countries outside of China [12] . (Figure 1 ) At the meeting of the Emergency Committee of the WHO on 30 January, the novel coronavirus 2019 epidemic was declared a Public Health Emergency of International Concern (PHEIC) [11, 13] .", "(Figure 1 ) At the meeting of the Emergency Committee of the WHO on 30 January, the novel coronavirus 2019 epidemic was declared a Public Health Emergency of International Concern (PHEIC) [11, 13] . This newly recognized coronavirus, producing a disease that has been termed COVID-19, is rapidly spreading throughout China, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV.", "It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV. In order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy. Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women.", "Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. It is the most prevalent non-obstetric infectious condition that occurs during pregnancy [14] [15] [16] . In one study pneumonia was the 3rd most common cause of indirect maternal death [17] .", "In one study pneumonia was the 3rd most common cause of indirect maternal death [17] . Approximately 25 percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [16] . Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] .", "Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] . As with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [19] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [20] [21] [22] . This has been evident historically during previous epidemics.", "This has been evident historically during previous epidemics. The case fatality rate (CFR) for pregnant women infected with influenza during the 1918-1919 pandemic was 27%-even higher when exposure occurred during the 3rd trimester and upwards of 50% if pneumonia supervened [23] . During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] .", "During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] . The most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include This newly recognized coronavirus, producing a disease that has been termed COVID-19, is rapidly spreading throughout China, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV.", "It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV. In order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy. Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women.", "Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. It is the most prevalent non-obstetric infectious condition that occurs during pregnancy [14] [15] [16] . In one study pneumonia was the 3rd most common cause of indirect maternal death [17] .", "In one study pneumonia was the 3rd most common cause of indirect maternal death [17] . Approximately 25 percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [16] . Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] .", "Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] . As with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [19] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [20] [21] [22] . This has been evident historically during previous epidemics.", "This has been evident historically during previous epidemics. The case fatality rate (CFR) for pregnant women infected with influenza during the 1918-1919 pandemic was 27%-even higher when exposure occurred during the 3rd trimester and upwards of 50% if pneumonia supervened [23] . During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] .", "During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] . The most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include premature rupture of membranes (PROM) and preterm labor (PTL), intrauterine fetal demise (IUFD), intrauterine growth restriction (IUGR), and neonatal death [14] [15] [16] . The SARS epidemic began quietly at the turn of the 21st century.", "The SARS epidemic began quietly at the turn of the 21st century. In November 2002, a cook in Guangdong Province, China, died from an unidentified illness. He had worked at a restaurant in which meat from wild animals was served.", "He had worked at a restaurant in which meat from wild animals was served. On 27 November 2002 Chinese-language media and internet reports were picked up by Canada's Global Public Health Intelligence Network (GPHIN) that indicated a flu-like illness was occurring in China [25, 26] . Unfortunately, the reports were not translated, and China failed to report the occurrence of this illness to the World Health Organization (WHO) until February 2003.", "Unfortunately, the reports were not translated, and China failed to report the occurrence of this illness to the World Health Organization (WHO) until February 2003. The disease spread to other countries where it primarily infected healthcare workers. One of these was Dr. Carlo Urbani, a WHO physician investigating a patient with the new disease in Hanoi.", "One of these was Dr. Carlo Urbani, a WHO physician investigating a patient with the new disease in Hanoi. He recognized that the pneumonia was probably caused by a new, highly infectious agent, and rapidly notified the WHO. He contracted the SARS-CoV while there, became febrile and later died after traveling to Thailand to attend a conference.", "He contracted the SARS-CoV while there, became febrile and later died after traveling to Thailand to attend a conference. On 12 March 2003, WHO issued a global alert regarding the disease that was occurring primarily among health care workers in Hanoi, Vietnam and Hong Kong. The disease continued to spread, and by 31 July 2003 there were 8422 probable cases, leading to 916 deaths in 29 countries, with the majority of cases occurring in mainland China and Hong Kong.", "The disease continued to spread, and by 31 July 2003 there were 8422 probable cases, leading to 916 deaths in 29 countries, with the majority of cases occurring in mainland China and Hong Kong. Approximately 30% of infections occurred in healthcare workers. By the termination of the epidemic the global CFR was 11% [27] .", "By the termination of the epidemic the global CFR was 11% [27] . Although there were relatively few documented cases of SARS occurring during pregnancy, several case reports and small clinical studies have described the clinical effects in pregnant women and their infants. In reviewing these reports describing pregnant women with SARS in China it is possible, and perhaps even probable, that some of the same patients were included in more than one publication.", "In reviewing these reports describing pregnant women with SARS in China it is possible, and perhaps even probable, that some of the same patients were included in more than one publication. However, even if this is the case, there is no doubt that SARS coronavirus infection was found to be associated with severe maternal illness, maternal death, and spontaneous abortion [19, [28] [29] [30] [31] . Martha Anker, an expert in statistics formerly with the WHO and the University of Massachusetts, estimated that more than 100 cases of SARS-CoV infection occurred in pregnant women, which warrants closer inspection [27] .", "Martha Anker, an expert in statistics formerly with the WHO and the University of Massachusetts, estimated that more than 100 cases of SARS-CoV infection occurred in pregnant women, which warrants closer inspection [27] . The clinical outcomes among pregnant women with SARS in Hong Kong were worse than those occurring in infected women who were not pregnant [32] . Wong et al.", "Wong et al. [29] evaluated the obstetrical outcomes from a cohort of pregnant women who developed SARS in Hong Kong during the period of 1 February to 31 July 2003. Four of the 7 women (57%) that presented during the 1st trimester sustained spontaneous miscarriages, likely a result of the hypoxia that was caused by SARS-related acute respiratory distress.", "Four of the 7 women (57%) that presented during the 1st trimester sustained spontaneous miscarriages, likely a result of the hypoxia that was caused by SARS-related acute respiratory distress. Among the 5 women who presented after 24 weeks gestation, 4 had preterm deliveries (80%). A case-control study to determine the effects of SARS on pregnancy compared 10 pregnant and 40 non-pregnant women with the infection at the Princess Margaret Hospital in Hong Kong [27, 33] .", "A case-control study to determine the effects of SARS on pregnancy compared 10 pregnant and 40 non-pregnant women with the infection at the Princess Margaret Hospital in Hong Kong [27, 33] . There were 3 deaths among the pregnant women with SARS (maternal mortality rate of 30%) and no deaths in the non-pregnant group of infected women (P = 0.006). Renal failure (P = 0.006) and disseminated intravascular coagulopathy (P = 0.006) developed more frequently in pregnant SARS patients when compared with the non-pregnant SARS group.", "Renal failure (P = 0.006) and disseminated intravascular coagulopathy (P = 0.006) developed more frequently in pregnant SARS patients when compared with the non-pregnant SARS group. Six pregnant women with SARS required admission to the intensive care unit (ICU) (60%) and 4 required endotracheal intubation (40%), compared with a 12.5% intubation rate (P = 0.065) and 17.5% ICU admission rate (P = 0.012) in the non-pregnant group. Maxwell et al.", "Maxwell et al. [32] reported 7 pregnant women infected with SARS-CoV who were followed at a designated SARS unit-2 of the 7 died (CFR of 28%), and 4 (57%) required ICU hospitalization and mechanical ventilation. In contrast, the mortality rate was less than 10% and mechanical ventilation rate less than 20% among non-pregnant, age-matched counterparts who were not infected with SARS-CoV.", "In contrast, the mortality rate was less than 10% and mechanical ventilation rate less than 20% among non-pregnant, age-matched counterparts who were not infected with SARS-CoV. Two women with SARS recovered and maintained their pregnancy but had infants with IUGR. Among the live newborn infants, none had clinical or laboratory evidence for SARS-CoV infection.", "Among the live newborn infants, none had clinical or laboratory evidence for SARS-CoV infection. The new mothers who had developed SARS were advised not to breastfeed to prevent possible vertical transmission of the virus. Zhang et al. [34] described SARS-CoV infections in 5 primagravidas from Guangzhou, China at the height of the SARS epidemic.", "[34] described SARS-CoV infections in 5 primagravidas from Guangzhou, China at the height of the SARS epidemic. Two of the mothers became infected in the 2nd trimester, and 3 developed infection in the 3rd trimester. Two of the pregnant women had hospital-acquired SARS infections, and the other 3 were community-acquired.", "Two of the pregnant women had hospital-acquired SARS infections, and the other 3 were community-acquired. All 5 pregnant women had fever and abnormal chest radiographs; 4 had cough; 4 developed hypoalbuminemia; 3 had elevated alanine aminotransferase levels (ALT), 3 had chills or rigor, 2 had decreased lymphocytes, and 2 had decreased platelets. One pregnant woman required intensive care, but all recovered and there were no maternal deaths.", "One pregnant woman required intensive care, but all recovered and there were no maternal deaths. The 5 infants were clinically evaluated, and none had evidence of SARS. Two pregnant women with SARS were reported from the United States. In a detailed case report, Robertson et al.", "In a detailed case report, Robertson et al. [35] described a 36-year-old pregnant woman with an intermittent cough of approximately 10 days duration and no fever. While travelling in Hong Kong during the 2003 epidemic, she was exposed at her hotel to a person subsequently known to be infected with SARS-CoV.", "While travelling in Hong Kong during the 2003 epidemic, she was exposed at her hotel to a person subsequently known to be infected with SARS-CoV. At 19 weeks gestation she developed fever, anorexia, headache, increasing cough, weakness, and shortness of breath. Upon returning to the United States she was hospitalized with pneumonia.", "Upon returning to the United States she was hospitalized with pneumonia. Obstetrical ultrasounds revealed a low-lying placenta (placenta previa) but were otherwise normal. Following her discharge home and clinical recovery, she was found to have antibodies to SARS-CoV.", "Following her discharge home and clinical recovery, she was found to have antibodies to SARS-CoV. She underwent cesarean section at 38 weeks gestation because of the placenta previa and a healthy baby girl was delivered [35, 36] . The placenta was interpreted as being normal.", "The placenta was interpreted as being normal. At 130 days post-maternal illness, maternal serum and whole blood, swabs from maternal nasopharynx and rectum, post-delivery placenta, umbilical cord blood, amniotic fluid, and breast milk were collected for analysis-no viral RNA was detected in specimens tested by reverse transcriptase polymerase chain reaction (RT-PCR). Antibodies to SARS-CoV were detected from maternal serum, umbilical cord blood, and breast milk by enzyme immunoassay (EIA) and indirect immunofluorescence assay.", "Antibodies to SARS-CoV were detected from maternal serum, umbilical cord blood, and breast milk by enzyme immunoassay (EIA) and indirect immunofluorescence assay. No clinical specimens (except for cord blood) were available for testing from the infant. The second case in the USA occurred in a 38-year-old woman who had travelled to Hong Kong at 7 weeks gestation where she was exposed to SARS-CoV in the same hotel as the aforementioned American woman [37] .", "The second case in the USA occurred in a 38-year-old woman who had travelled to Hong Kong at 7 weeks gestation where she was exposed to SARS-CoV in the same hotel as the aforementioned American woman [37] . Following her return to the United States, her husband developed the clinical onset of SARS, and 6 days later she became ill with fever, myalgia, chills, headache, coryza, and a productive cough with shortness of breath and wheezing. Following her hospitalization for SARS she recovered, serum samples taken on days 28 and 64 post-onset of illness were positive for antibodies to SARS-CoV by enzyme immunoassay and immunofluorescent assays.", "Following her hospitalization for SARS she recovered, serum samples taken on days 28 and 64 post-onset of illness were positive for antibodies to SARS-CoV by enzyme immunoassay and immunofluorescent assays. Her pregnancy continued and was unremarkable except for developing elevated glucose levels. A cesarean section that was performed at 36 weeks gestation due to preterm rupture of membranes and fetal distress resulted in a healthy baby boy.", "A cesarean section that was performed at 36 weeks gestation due to preterm rupture of membranes and fetal distress resulted in a healthy baby boy. At the time of delivery, the mother's serum samples were positive for antibodies to SARS-CoV, but samples taken of umbilical cord blood and placenta were negative. Breast milk sampled 12 and 30 days after delivery were also negative for SARS-CoV antibodies.", "Breast milk sampled 12 and 30 days after delivery were also negative for SARS-CoV antibodies. Specimens evaluated from maternal blood, stool, and nasopharynx samples, as well as umbilical cord blood of the infant, were all negative for coronavirus RNA by RT-PCR. Neonatal stool samples obtained on days-of-life 12 and 30 were also negative for viral RNA.", "Neonatal stool samples obtained on days-of-life 12 and 30 were also negative for viral RNA. From Canada, Yudin et al. [38] reported a 33-year-old pregnant woman who was admitted to the hospital at 31 weeks gestation with a fever, dry cough, and abnormal chest radiograph demonstrating patchy infiltrates.", "[38] reported a 33-year-old pregnant woman who was admitted to the hospital at 31 weeks gestation with a fever, dry cough, and abnormal chest radiograph demonstrating patchy infiltrates. She had acquired SARS from contact with an infected family member. Following a 21-day stay in the hospital, during which she did not require ventilatory support, her convalescent antibody titers were positive for coronavirus infection.", "Following a 21-day stay in the hospital, during which she did not require ventilatory support, her convalescent antibody titers were positive for coronavirus infection. She had a normal labor and delivery and her newborn girl had no evidence of infection. In a study of 5 liveborn neonates who were delivered to women infected with SARS-CoV during the Hong Kong epidemic, results from multiple tests-including serial RT-PCR assays, viral culture, and paired neonatal serological titers-were negative for SARS-CoV [39] .", "In a study of 5 liveborn neonates who were delivered to women infected with SARS-CoV during the Hong Kong epidemic, results from multiple tests-including serial RT-PCR assays, viral culture, and paired neonatal serological titers-were negative for SARS-CoV [39] . None of the 5 neonates developed any clinical signs or symptoms of respiratory infection or compromise. Fortunately, there were no cases of vertical transmission identified among pregnant women infected with SARS-CoV during the 2002-2003 Asian epidemic [27, 30, 31, 39, 40] , and with the exception of a small cluster of cases that recurred in late 2003, no new cases of SARS have occurred.", "Fortunately, there were no cases of vertical transmission identified among pregnant women infected with SARS-CoV during the 2002-2003 Asian epidemic [27, 30, 31, 39, 40] , and with the exception of a small cluster of cases that recurred in late 2003, no new cases of SARS have occurred. In the only reported study of the placental pathology of mothers with SARS, Ng et al. [41] reported the findings from 7 pregnant women infected with SARS-CoV.", "[41] reported the findings from 7 pregnant women infected with SARS-CoV. In the case of 2 women who were convalescing from SARS-CoV infection during the 1st trimester of pregnancy, the placentas were found to be normal. Three placentas were delivered from pregnancies in which the mothers had acute SARS-CoV infection-these were abnormal and demonstrated increased subchorionic and intervillous fibrin, a finding that can be associated with abnormal maternal blood flow to the placenta.", "Three placentas were delivered from pregnancies in which the mothers had acute SARS-CoV infection-these were abnormal and demonstrated increased subchorionic and intervillous fibrin, a finding that can be associated with abnormal maternal blood flow to the placenta. In the placentas of 2 women who were convalescing from SARS-CoV infection in the 3rd trimester of pregnancy the placentas were highly abnormal. They showed extensive fetal thrombotic vasculopathy with areas of avascular chorionic villi-chronic findings of fetal vascular malperfusion.", "They showed extensive fetal thrombotic vasculopathy with areas of avascular chorionic villi-chronic findings of fetal vascular malperfusion. These 2 pregnancies also were complicated by oligohydramnios and had poor obstetrical outcomes-both infants had developed IUGR. It is interesting that villitis, the microscopic finding of inflammation of the chorionic villi that is the histologic hallmark of many maternal hematogenous infections that are transmitted through the placenta to the fetus, was not identified in any of these placentas.", "It is interesting that villitis, the microscopic finding of inflammation of the chorionic villi that is the histologic hallmark of many maternal hematogenous infections that are transmitted through the placenta to the fetus, was not identified in any of these placentas. Similar to other coronavirus infections, SARS-CoV is easily spread from person-to-person via respiratory droplets and secretions as well as through nosocomial contacts [42, 43] . In addition to transmission of SARS-CoV through natural aerosols from infected patients, it was found that in Hong Kong the SARS-CoV could also be transmitted by mechanical aerosols [44] .", "In addition to transmission of SARS-CoV through natural aerosols from infected patients, it was found that in Hong Kong the SARS-CoV could also be transmitted by mechanical aerosols [44] . Environmental factors had an important role when it was discovered that during the Amoy Gardens housing estate outbreak as many as two-thirds of infected persons had diarrhea, SARS-CoV was excreted in their stools, and that aerosols arising from the flushing of toilets could transmit the virus [44] . Healthcare facilities were also an important source of new SARS infections during the 2002-2003 epidemic, and healthcare workers were also at high risk for acquiring the infection.", "Healthcare facilities were also an important source of new SARS infections during the 2002-2003 epidemic, and healthcare workers were also at high risk for acquiring the infection. In order to address the safety issues for the obstetrical management and delivery of pregnant women with SARS, guidelines were prepared by the Canadian Task Force on Preventive Health Care and the Society of Obstetricians and Gynaecologists of Canada [45] . These recommendations include:\n\n1.", "These recommendations include:\n\n1. \"All hospitals should have infection control systems in place to ensure that alerts regarding changes in exposure risk factors for SARS or other potentially serious communicable diseases are conveyed promptly to clinical units, including the labour and delivery unit. At times of SARS outbreaks, all pregnant patients being assessed or admitted to the hospital should be screened for symptoms of and risk factors for SARS.", "At times of SARS outbreaks, all pregnant patients being assessed or admitted to the hospital should be screened for symptoms of and risk factors for SARS. Upon arrival in the labour triage unit, pregnant patients with suspected and probable SARS should be placed in a negative pressure isolation room with at least 6 air exchanges per hour. All labour and delivery units caring for suspected and probable SARS should have available at least one room in which patients can safely labour and deliver while in need of airborne isolation.", "All labour and delivery units caring for suspected and probable SARS should have available at least one room in which patients can safely labour and deliver while in need of airborne isolation. If possible, labour and delivery (including operative delivery or Caesarean section) should be managed in a designated negative pressure isolation room, by designated personnel with specialized infection control preparation and protective gear. 5.", "5. Either regional or general anaesthesia may be appropriate for delivery of patients with SARS. Neonates of mothers with SARS should be isolated in a designated unit until the infant has been well for 10 days, or until the mother's period of isolation is complete. The mother should not breastfeed during this period. 7.", "The mother should not breastfeed during this period. 7. A multidisciplinary team, consisting of obstetricians, nurses, pediatricians, infection control specialists, respiratory therapists, and anaesthesiologists, should be identified in each unit and be responsible for the unit organization and implementation of SARS management protocols. 8.", "8. Staff caring for pregnant SARS patients should not care for other pregnant patients. Staff caring for pregnant SARS patients should be actively monitored for fever and other symptoms of SARS. Such individuals should not work in the presence of any SARS symptoms within 10 days of exposure to a SARS patient. 9.", "9. All health care personnel, trainees, and support staff should be trained in infection control management and containment to prevent spread of the SARS virus. 10.", "10. Regional health authorities in conjunction with hospital staff should consider designating specific facilities or health care units, including primary, secondary, or tertiary health care centers, to care for patients with SARS or similar illnesses.\" Middle East respiratory syndrome (MERS) was first reported in September 2012 in Saudi Arabia, following isolation of MERS-CoV from a male patient who died months earlier from severe pneumonia and multiple organ failure [1] .", "Middle East respiratory syndrome (MERS) was first reported in September 2012 in Saudi Arabia, following isolation of MERS-CoV from a male patient who died months earlier from severe pneumonia and multiple organ failure [1] . In the 8 years since then, there have been more than 2494 confirmed cases of MERS resulting in upwards of 858 deaths globally [46] . While 27 countries have reported cases of MERS, approximately 80% of confirmed cases originated in Saudi Arabia [47] .", "While 27 countries have reported cases of MERS, approximately 80% of confirmed cases originated in Saudi Arabia [47] . To date, all known cases of MERS can be linked to travel or residence in countries along the Arabian Peninsula-that is, Bahrain; Iraq; Iran; Israel, the West Bank, and Gaza; Jordan; Kuwait; Lebanon; Oman; Qatar, Saudi Arabia; Syria; the United Arab Emirates (UAE); and Yemen [48] . The largest documented outbreak outside of this region occurred in 2015 in the Republic of Korea, in which 186 infections occurred, resulting in 38 deaths [49] .", "The largest documented outbreak outside of this region occurred in 2015 in the Republic of Korea, in which 186 infections occurred, resulting in 38 deaths [49] . The index case in this outbreak reportedly returned from the Arabian Peninsula just prior to onset of illness [50] . MERS-CoV is characterized by sporadic zoonotic transmission events as well as spread between infected patients and close contacts (i.e., intra-familial transmission) [51] .", "MERS-CoV is characterized by sporadic zoonotic transmission events as well as spread between infected patients and close contacts (i.e., intra-familial transmission) [51] . Nosocomial outbreaks in health care settings-the result of poor infection control and prevention-are widely recognized as the hallmark of MERS [1] . Superspreading events have been recorded in healthcare settings in Jordan, Al Hasa, Jeddah, Abu Dhabi and South Korea [47, [52] [53] [54] [55] .", "Superspreading events have been recorded in healthcare settings in Jordan, Al Hasa, Jeddah, Abu Dhabi and South Korea [47, [52] [53] [54] [55] . Like other coronaviruses, MERS-CoV can be spread through person-to-person contact, likely via infected respiratory secretions [48] . Transmission dynamics, however, are otherwise poorly understood [1] .", "Transmission dynamics, however, are otherwise poorly understood [1] . Bats are believed to be the natural reservoir of MERS-CoV, and dromedary camels can have the virus and have been suggested as possible intermediary hosts as well as a source of infection to humans [2, 56, 57] . There are no clinical or serological reports of perinatal transmission of MERS, though vertical transmission has been reported for non-coronavirus respiratory viruses including influenza and respiratory syncytial virus (RSV) [58] .", "There are no clinical or serological reports of perinatal transmission of MERS, though vertical transmission has been reported for non-coronavirus respiratory viruses including influenza and respiratory syncytial virus (RSV) [58] . Researchers have not yet discovered ongoing transmission of MERS-CoV within communities outside of health care settings. The clinical presentation of MERS varies from asymptomatic to severe pneumonia with acute respiratory distress syndrome (ARDS), septic shock, and multiple organ failure, often resulting in death.", "The clinical presentation of MERS varies from asymptomatic to severe pneumonia with acute respiratory distress syndrome (ARDS), septic shock, and multiple organ failure, often resulting in death. Most patients with MERS develop severe acute respiratory illness accompanied by fever, cough, and shortness of breath [50] . Progression to pneumonia is swift-usually within the first week -and at least one-third of patients also present with gastrointestinal symptoms [1] .", "Progression to pneumonia is swift-usually within the first week -and at least one-third of patients also present with gastrointestinal symptoms [1] . MERS progresses much more rapidly to respiratory failure and has a higher case fatality rate than SARS [1] . Unlike SARS, however, infection with MERS-CoV is generally mild in healthy individuals but more severe in immunocompromised patients and people with underlying comorbidities [1] .", "Unlike SARS, however, infection with MERS-CoV is generally mild in healthy individuals but more severe in immunocompromised patients and people with underlying comorbidities [1] . The overall CFR of MERS is approximately 34.4% [46] . Most fatalities have been associated with pre-existing medical conditions like chronic lung disease, diabetes, and renal failure, as well as weakened immune systems [59] , making such individuals high risk.", "Most fatalities have been associated with pre-existing medical conditions like chronic lung disease, diabetes, and renal failure, as well as weakened immune systems [59] , making such individuals high risk. As a result of the immunological changes that occur during pregnancy, women who are pregnant are included in this high-risk group. Pregnant women may develop severe disease and fatal maternal and/or fetal outcomes as a result of MERS-CoV infection; however, little is known of the pathophysiology of this infection during pregnancy.", "Pregnant women may develop severe disease and fatal maternal and/or fetal outcomes as a result of MERS-CoV infection; however, little is known of the pathophysiology of this infection during pregnancy. Limited data exists on the prevalence and clinical features of MERS during pregnancy, birth, and the postnatal period. It is likely, however, that the immunological changes that normally occur in pregnancy may alter susceptibility to the MERS-CoV and the severity of clinical illness [60] .", "It is likely, however, that the immunological changes that normally occur in pregnancy may alter susceptibility to the MERS-CoV and the severity of clinical illness [60] . Pregnant women infected with SARS-CoV, a related coronavirus, appear to have increased morbidity and mortality when compared to non-pregnant women, suggesting that MERS-CoV could also lead to severe clinical outcomes in pregnancy. To date, however, very few pregnancy-associated cases (n = 11) have been documented, with 91% having adverse clinical outcomes.", "To date, however, very few pregnancy-associated cases (n = 11) have been documented, with 91% having adverse clinical outcomes. Between November 2012 and February 2016, there were 1308 cases of MERS reported by the Saudi Arabia Ministry of Health (MoH). Of these, 5 patients were pregnant, according to a retrospective study by Assiri et al.", "Of these, 5 patients were pregnant, according to a retrospective study by Assiri et al. [47] , and all resulted in adverse outcomes. Patient ages ranged from 27 to 34 years, with occurrence of exposure in either the 2nd or 3rd trimester. All 5 cases received intensive care.", "All 5 cases received intensive care. Two women died and there were 2 cases of perinatal death-1 stillbirth and 1 neonatal death shortly after emergency cesarean section. These instances of severe maternal and perinatal outcomes are consistent with other reports of MERS-CoV infection in pregnant women, as well as outcomes associated with SARS-CoV infection.", "These instances of severe maternal and perinatal outcomes are consistent with other reports of MERS-CoV infection in pregnant women, as well as outcomes associated with SARS-CoV infection. The authors of the retrospectives study concede that unreported cases of MERS in pregnancy are likely due to lack of routine pregnancy testing [47] . They conclude that pregnancy testing for women of reproductive age should be considered for those who test positive for MERS-CoV, to contribute to overall understanding of pathogenesis and epidemiological risk.", "They conclude that pregnancy testing for women of reproductive age should be considered for those who test positive for MERS-CoV, to contribute to overall understanding of pathogenesis and epidemiological risk. Additionally, 2 of the 5 patients were healthcare workers, which corresponds with existing knowledge of higher risk of exposure to MERS-CoV in healthcare settings. In a separate case report of MERS occurring in pregnancy, Alserehi et al.", "In a separate case report of MERS occurring in pregnancy, Alserehi et al. [58] described a 33-year-old critical care nurse who became infected during the 3rd trimester in the midst of a large hospital outbreak. In the days following hospital admission, she developed respiratory failure necessitating mechanical ventilation and administration of dexamethasone as prophylaxis for the fetus.", "In the days following hospital admission, she developed respiratory failure necessitating mechanical ventilation and administration of dexamethasone as prophylaxis for the fetus. Following an emergency cesarean section at 32 weeks gestation, she was transferred to the intensive care unit (ICU) and later recovered. The preterm but otherwise healthy infant was kept in the neonatal unit for observation and later released along with his mother.", "The preterm but otherwise healthy infant was kept in the neonatal unit for observation and later released along with his mother. In contrast to other reported cases, this patient had a successful outcome, perhaps due to the timing of MERS-CoV exposure, her young age, the use of steroids, and differences in immune response. Alfaraj et al.", "Alfaraj et al. [61] described 2 cases of maternal infection with MERS-CoV at the Prince Mohammed Bin Abdulaziz Hospital (PMAH) in Saudi Arabia. Maternal infection in both cases was confirmed by nasopharyngeal swab testing by RT-PCR. One patient was a 29-year-old woman at 6 weeks gestation with no underlying medical conditions.", "One patient was a 29-year-old woman at 6 weeks gestation with no underlying medical conditions. The second patient, a 39-year-old at 24 weeks gestation, had several comorbidities, including end stage renal disease, hypertension, and hemodialysis. This woman presented to the hospital after contact with a MERS-CoV-infected person during an active outbreak.", "This woman presented to the hospital after contact with a MERS-CoV-infected person during an active outbreak. Both patients later tested negative for MERS-CoV and were subsequently discharged. The younger patient delivered a healthy, full-term infant. The status of the other delivery is unknown. Neither fetus was tested for MERS-CoV. According to Payne et al.", "Neither fetus was tested for MERS-CoV. According to Payne et al. [62] , epidemiologic investigation of the 2012 MERS outbreak in Zarqa, Jordan, revealed that a 2nd trimester stillbirth (5 months gestational age) had occurred as a result of maternal exposure to MERS-CoV.", "[62] , epidemiologic investigation of the 2012 MERS outbreak in Zarqa, Jordan, revealed that a 2nd trimester stillbirth (5 months gestational age) had occurred as a result of maternal exposure to MERS-CoV. The mother experienced fever, fatigue, headache and cough, concurrently with vaginal bleeding and abdominal pain. On the 7th day of symptoms, she had a fetal death.", "On the 7th day of symptoms, she had a fetal death. The mother was confirmed to have antibody to MERS-CoV, and she self-reported having had unprotected contact with family members who later tested positive for the virus. This was the first documented occurrence of stillbirth during maternal infection with MERS-CoV.", "This was the first documented occurrence of stillbirth during maternal infection with MERS-CoV. On 24 November 2013, a 32-year-old pregnant woman in the United Arab Emirates (UAE) developed ARDS following admission to the ICU after suspected community-acquired pneumonia advanced to respiratory failure and hypotension [60] . Later that day, her baby was delivered by caesarean section and subsequent Apgar scores were within healthy range.", "Later that day, her baby was delivered by caesarean section and subsequent Apgar scores were within healthy range. The next day, RT-PCR evaluation revealed that the mother was positive for MERS-CoV. Despite rigorous intervention, including oral ribavirin-peginterferon-α therapy and ventilator support, the woman continued to deteriorate, developed septic shock, and died.", "Despite rigorous intervention, including oral ribavirin-peginterferon-α therapy and ventilator support, the woman continued to deteriorate, developed septic shock, and died. While the outcome for this mother was fatal, Malik et al. noted that virus shedding ceased during therapy with ribavirin and peginterferon-α and radiographic evidence indicated clinical improvement before her death [58] .", "noted that virus shedding ceased during therapy with ribavirin and peginterferon-α and radiographic evidence indicated clinical improvement before her death [58] . More research is needed to determine safety, efficacy, and dosage of these therapies in the general population but also in pregnant women. While few data exist on the effects of these treatments in pregnant humans, ribavirin is generally contraindicated during pregnancy [58] .", "While few data exist on the effects of these treatments in pregnant humans, ribavirin is generally contraindicated during pregnancy [58] . Outside of the Middle East the only confirmed case of MERS in pregnancy occurred in 2015 in South Korea. Jeong et al.", "Jeong et al. [49] reported that a 39-year-old patient was exposed during the 3rd trimester following contact with a patient having MERS. Despite abrupt vaginal bleeding and rupture of membranes, the patient recovered fully and delivered a healthy infant at 37 weeks and 5 days gestation.", "Despite abrupt vaginal bleeding and rupture of membranes, the patient recovered fully and delivered a healthy infant at 37 weeks and 5 days gestation. Subsequent testing of the infant's blood did not detect any IgG, IgM, or IgA antibodies to MERS-CoV. The mean maternal age of the 11 confirmed maternal SARS cases described above was 33.2 years, with a mean gestational age of 26.3 weeks.", "The mean maternal age of the 11 confirmed maternal SARS cases described above was 33.2 years, with a mean gestational age of 26.3 weeks. The source of infection in 2 of the cases was attributed to contact with family members who tested positive for MERS-CoV, unknown in 3 cases, likely due to animal exposure in 1 case, and 6 were healthcare-associated (2 of these patients were healthcare workers). Six patients required intensive care and 3 died.", "Six patients required intensive care and 3 died. Of those who died, 2 were exposed to MERS-CoV in the 3rd trimester, and 1 was exposed during the 2nd trimester. The infant death rate for all 11 cases was 27%.", "The infant death rate for all 11 cases was 27%. Fetal survival did not appear to correlate with the timing of maternal infection and gestational age; however, more data are needed to draw conclusions about this relationship. According to Alfaraj et al.", "According to Alfaraj et al. [61] , the CFR for the 11 infected women-also 27%-was not statistically different from the overall CFR of MERS in the general population (35%) (P = 0.75). Only 1 case resulted in both maternal and fetal death.", "Only 1 case resulted in both maternal and fetal death. Similar to SARS in pregnancy, more research is needed to understand the pathogenesis and epidemiology of MERS in pregnancy including the relationship between the timing of maternal infection, gestational age of the fetus, the effects of comorbid factors, and the occurrence of adverse outcomes. Few studies documented the presence of MERS-CoV antibodies in the umbilical cord or neonatal blood, making it difficult to assess perinatal transmission.", "Few studies documented the presence of MERS-CoV antibodies in the umbilical cord or neonatal blood, making it difficult to assess perinatal transmission. As such, future studies should involve the collection of samples from relevant specimens including amniotic fluid, placenta, and umbilical cord [49] . MERS prevention should be high priority for high-risk exposures such as healthcare workers, pregnant women and individuals working with camels, camel meat-milk processors and in abattoirs [57] .", "MERS prevention should be high priority for high-risk exposures such as healthcare workers, pregnant women and individuals working with camels, camel meat-milk processors and in abattoirs [57] . Since 2013, the Saudi Arabia MoH has recommended that pregnant women postpone travel to Saudi Arabia for the Hajj and Umrah [47] . To further reduce risk of exposure among pregnant women, additional measures such as avoiding contact with camels and sick persons-particularly in healthcare settings-are also recommended.", "To further reduce risk of exposure among pregnant women, additional measures such as avoiding contact with camels and sick persons-particularly in healthcare settings-are also recommended. Pregnant women who present with symptoms of pneumonia, influenza-like illness (ILI), or sepsis on the Arabian Peninsula may also benefit from MERS-CoV screening to expedite early diagnosis and improve disease management [60] . While multiple agents have been used to treat MERS, none have been tested in large clinical studies.", "While multiple agents have been used to treat MERS, none have been tested in large clinical studies. Available data are limited to the use of combination therapies of interferon and other agents in case reports and case series [63] . A prospective or randomized study may prove difficult given the sporadic nature of MERS-CoV outbreaks.", "A prospective or randomized study may prove difficult given the sporadic nature of MERS-CoV outbreaks. Due to a gap in research on the treatment of MERS in pregnancy, there are no therapeutic options currently recommended for pregnant women [58] . Therapies under development and testing may be considered inappropriate for pregnant women due to the unknown potential for teratogenic effects.", "Therapies under development and testing may be considered inappropriate for pregnant women due to the unknown potential for teratogenic effects. For example, during the 2003 SARS outbreak, ribavirin was administered to pregnant women with severe cases of the disease, but ribavirin therapy has been documented to increase the risk of teratogenic effects in newborns [58] . The Alphacoronaviruses HCoV 229E and NL63, as well as the Betacoronaviruses HKU 1 and OC43, can infect humans and cause the common cold.", "The Alphacoronaviruses HCoV 229E and NL63, as well as the Betacoronaviruses HKU 1 and OC43, can infect humans and cause the common cold. In order to investigate the potential maternal-fetal transmission of human coronaviruses during pregnancy, Gagneur et al. [64, 65] evaluated 3 types of maternal-infant paired specimens that included maternal vaginal and respiratory specimens that were obtained during labor, as well as gastric samples from the newborn infants.", "[64, 65] evaluated 3 types of maternal-infant paired specimens that included maternal vaginal and respiratory specimens that were obtained during labor, as well as gastric samples from the newborn infants. These specimens were evaluated for the presence of HCoV 229E, OC-43, NL63 and HKU 1 using RT-PCR methodology. Between the period from July 2003 to August 2005 the authors examined 159 mother-infant dyads.", "Between the period from July 2003 to August 2005 the authors examined 159 mother-infant dyads. Human coronaviruses were identified in 12 samples (HCoV 229E: 11; HKU 1 : 1) from 7 mother-child pairs. In 3 mother-infant dyads only maternal respiratory samples were positive; in 2 other pairs all 3 of the samples tested positive for human coronavirus; in 1 case only the maternal vaginal and newborn gastric samples were positive; and in another case the maternal vaginal sample alone was positive.", "In 3 mother-infant dyads only maternal respiratory samples were positive; in 2 other pairs all 3 of the samples tested positive for human coronavirus; in 1 case only the maternal vaginal and newborn gastric samples were positive; and in another case the maternal vaginal sample alone was positive. There were no signs of clinical infection in any of the 3 neonates that had positive gastric samples for human coronavirus. It is beyond the scope of this communication to discuss the various technical challenges inherent in developing a safe and efficacious vaccine for coronavirus infections in humans.", "It is beyond the scope of this communication to discuss the various technical challenges inherent in developing a safe and efficacious vaccine for coronavirus infections in humans. There are clearly challenges to this endeavor-protective antibodies to coronaviruses are not long-lasting, tissue damage has been reported to occur as a result of exposure to SARS-CoV, development of animal models that closely resemble human infection are limited, and the extensive time and expense necessary to perform clinical trials in humans, to name a few [66] [67] [68] . It is vitally important that pregnant women be considered in the design, clinical trial, and implementation of vaccine candidates for 2019-nCoV.", "It is vitally important that pregnant women be considered in the design, clinical trial, and implementation of vaccine candidates for 2019-nCoV. In examining the history of vaccine design, it is clear that the needs of pregnant women have rarely been prioritized in either the preclinical development or the clinical trial phases of production. Today, pregnant women are usually excluded from experimental trial of drugs and vaccines that do not target obstetric conditions [69] .", "Today, pregnant women are usually excluded from experimental trial of drugs and vaccines that do not target obstetric conditions [69] . Excluding pregnant women and their infants from participation in vaccine development and implementation undermines ethical principles of justice-fairness, equity, and maximization of benefit-and potentially places their health at risk during outbreaks and other health emergencies [69] [70] [71] . On 23 January 2020 the Coalition for Epidemic Preparedness Innovations (CEPI) announced three programs to develop a vaccine against the novel Wuhan coronavirus.", "On 23 January 2020 the Coalition for Epidemic Preparedness Innovations (CEPI) announced three programs to develop a vaccine against the novel Wuhan coronavirus. The Chief Executive Officer of CEPI, Richard Hatchett, said [72] :\n\n\"Given the rapid global spread of the nCoV-2019 virus the world needs to act quickly and in unity to tackle this disease. Our intention with this work is to leverage our work on the MERS coronavirus and rapid response platforms to speed up vaccine development.\"", "Our intention with this work is to leverage our work on the MERS coronavirus and rapid response platforms to speed up vaccine development.\" The novel coronavirus is the first epidemic disease to emerge since the formation of CEPI in Davos in 2017. CEPI was created with the express intent to enable speedy research and development of vaccines against emerging pathogens.", "CEPI was created with the express intent to enable speedy research and development of vaccines against emerging pathogens. In May 2017, WHO released the Target Product Profile (TPP) for MERS-CoV vaccines, following the prioritization of MERS-CoV as one of eight priority pathogens for prevention of epidemics [73] . CEPI and partners aim to use existing platforms-that is, the existing \"backbone\" that can be adapted for use against new pathogens-that are currently in preclinical development for MERS-CoV vaccine candidates.", "CEPI and partners aim to use existing platforms-that is, the existing \"backbone\" that can be adapted for use against new pathogens-that are currently in preclinical development for MERS-CoV vaccine candidates. Following the WHO declaration on 30 January that the current 2019-nCoV outbreak is a public health emergency of international concern (PHEIC), global health organizations and researchers will be further mobilized-bolstered by new mechanisms for action and greater resources-to stop the spread of disease. A critical question that must be answered at this stage-with a clear view of the potential deleterious effects of a new coronavirus in pregnancy-is will maternal immunization be a priority in research and development?", "A critical question that must be answered at this stage-with a clear view of the potential deleterious effects of a new coronavirus in pregnancy-is will maternal immunization be a priority in research and development? As of the PHEIC declaration, 12 groups have announced that they are developing new vaccines against 2019-nCoV and seven others announced initiatives to develop new therapies [74] . Safe testing of experimental vaccines in a pregnant population is difficult and, as a result, vaccines are not typically developed with pregnant women in mind.", "Safe testing of experimental vaccines in a pregnant population is difficult and, as a result, vaccines are not typically developed with pregnant women in mind. To date, very few clinical trials for vaccines have proactively included pregnant women [75] , and the exclusion of pregnant and lactating women from receiving the rVSV-ZEBOV vaccine through 3 Ebola virus epidemics serves as a recent example [69] [70] [71] . Given the potential severity in pregnancy, as demonstrated by this review of maternal infections of SARS and MERS, women who are pregnant should be considered a priority population in all efforts to prepare for and prevent infection by novel coronaviruses.", "Given the potential severity in pregnancy, as demonstrated by this review of maternal infections of SARS and MERS, women who are pregnant should be considered a priority population in all efforts to prepare for and prevent infection by novel coronaviruses. On 5 February 2020 it was reported by multiple media outlets that a newborn infant delivered during the epidemic in Wuhan had tested positive for 2019-nCoV at the Wuhan Children's Hospital in Hubei Province 30 hours following its birth. According to the official Xinhua news agency, the infant was delivered on 2 February to a mother who had tested positive for the virus.", "According to the official Xinhua news agency, the infant was delivered on 2 February to a mother who had tested positive for the virus. Reports have stated that the infant had stable vital signs, no fever or cough, but had shortness of breath together with abnormal chest radiographs and abnormalities of liver function [76] [77] [78] . Dr. Zeng Lingkong, Chief Physician at the Neonatal Medicine Department of the hospital, said [78] , \"This reminds us to pay attention to mother-to-child being a possible route of coronavirus transmission\"\n\nThe hospital also provided information about a previous case of a baby that had been delivered on 13 January 2020.", "Dr. Zeng Lingkong, Chief Physician at the Neonatal Medicine Department of the hospital, said [78] , \"This reminds us to pay attention to mother-to-child being a possible route of coronavirus transmission\"\n\nThe hospital also provided information about a previous case of a baby that had been delivered on 13 January 2020. Following its birth, the infant's nanny was diagnosed with 2019-nCoV, and the mother was diagnosed days later [76] . On 29 January the baby began to develop symptoms.", "On 29 January the baby began to develop symptoms. According to Dr. Zeng Lingkong [76] , \"Whether it was the baby's nanny who passed the virus to the mother who passed it to the baby, we cannot be sure at the moment. But we can confirm that the baby was in close contact with patients infected with the new coronavirus, which says newborns can also be infected\"\n\nIn considering whether these and future cases of neonatal infection are acquired prior to delivery, it is important to remember that newborn infants can acquire an infection in other ways beyond intrauterine maternal-fetal transmission.", "But we can confirm that the baby was in close contact with patients infected with the new coronavirus, which says newborns can also be infected\"\n\nIn considering whether these and future cases of neonatal infection are acquired prior to delivery, it is important to remember that newborn infants can acquire an infection in other ways beyond intrauterine maternal-fetal transmission. In some cases, viral infection can be acquired when the infant passes through the birth canal during a vaginal delivery or through post-partum breast feeding, although these mechanisms would be highly unusual for a respiratory virus. Neonatal infection from respiratory viruses can occur after delivery through such mechanisms as inhalation of the agent through aerosols produced by coughing from the mother, relatives or healthcare workers or other sources in the hospital environment.", "Neonatal infection from respiratory viruses can occur after delivery through such mechanisms as inhalation of the agent through aerosols produced by coughing from the mother, relatives or healthcare workers or other sources in the hospital environment. Based upon past experience with pregnant women who developed MERS and SARS, and realizing that the numbers are limited, there has never been confirmed intrauterine coronavirus transmission from mother to fetus. Discussing the most recent baby to be diagnosed with the 2019-nCoV infection, Dr. Stephen Morse, an epidemiologist at the Mailman School of Public Health at Columbia University stated [77] , \"It's more likely that the baby contracted the virus from the hospital environment, the same way healthcare workers get infected by the patients they treat,\" \"It's quite possible that the baby picked it up very conventionally-by inhaling virus droplets that came from the mother coughing.\"", "Discussing the most recent baby to be diagnosed with the 2019-nCoV infection, Dr. Stephen Morse, an epidemiologist at the Mailman School of Public Health at Columbia University stated [77] , \"It's more likely that the baby contracted the virus from the hospital environment, the same way healthcare workers get infected by the patients they treat,\" \"It's quite possible that the baby picked it up very conventionally-by inhaling virus droplets that came from the mother coughing.\" And according to Dr. Paul Hunter, Professor of Medicine at the University of East Anglia [79] , \"As far as I am aware there is currently no evidence that the novel coronavirus can be transmitted in the womb. When a baby is born vaginally it is exposed to the mother's gut microbiome, therefore if a baby does get infected with coronavirus a few days after birth we currently cannot tell if the baby was infected in the womb or during birth.\"", "When a baby is born vaginally it is exposed to the mother's gut microbiome, therefore if a baby does get infected with coronavirus a few days after birth we currently cannot tell if the baby was infected in the womb or during birth.\" There is limited knowledge regarding coronavirus infections that occur during pregnancy-what is known has, for the most part, been the result of epidemics resulting from two different diseases, SARS and MERS. These previous experiences with coronavirus infections in pregnancy indicates that these agents are capable of causing adverse clinical outcomes including life-threatening maternal disease that in some cases requires hospitalization, intensive care and ventilatory support.", "These previous experiences with coronavirus infections in pregnancy indicates that these agents are capable of causing adverse clinical outcomes including life-threatening maternal disease that in some cases requires hospitalization, intensive care and ventilatory support. Both of these coronaviruses can result in maternal death in a small but significant number of cases, but the specific risk factors for a fatal outcome during pregnancy have not been clarified. Coronaviruses can also result in adverse outcomes for the fetus and infant including intrauterine growth restriction, preterm delivery, admission to the ICU, spontaneous abortion and perinatal death.", "Coronaviruses can also result in adverse outcomes for the fetus and infant including intrauterine growth restriction, preterm delivery, admission to the ICU, spontaneous abortion and perinatal death. Unlike some viral infections, notably Ebola virus [70] and Zika virus [80] , the likelihood of intrauterine maternal-fetal transmission of coronaviruses is low-there have been no documented cases of vertical transmission occurring with either SARS or MERS. It remains to be seen during the current Wuhan 2019-nCoV epidemic how this newly-emergent coronavirus affects pregnant women and their infants, as well as which factors may modulate obstetrical disease and outcomes including the timing of maternal coronavirus exposure by gestational age, the effects of medications or other treatment regimens, differences in host immune responses, occurrence of coexisting medical and obstetrical conditions, and other covariables.", "It remains to be seen during the current Wuhan 2019-nCoV epidemic how this newly-emergent coronavirus affects pregnant women and their infants, as well as which factors may modulate obstetrical disease and outcomes including the timing of maternal coronavirus exposure by gestational age, the effects of medications or other treatment regimens, differences in host immune responses, occurrence of coexisting medical and obstetrical conditions, and other covariables. However, pregnant women should be considered to be at high risk for developing severe infection during this current outbreak of 2019-nCoV. Additional clinical research on the treatment of SARS, MERS, and the new coronavirus 2019-nCoV is necessary if we are to understand the potential risks and benefits of novel therapies and new vaccines in pregnancy.", "Additional clinical research on the treatment of SARS, MERS, and the new coronavirus 2019-nCoV is necessary if we are to understand the potential risks and benefits of novel therapies and new vaccines in pregnancy. This research will be critical in improving the care, and even saving the lives, of pregnant women in the current as well as future outbreaks." ]

[ "In early December 2019 a cluster of cases of pneumonia of unknown cause was identified in Wuhan, a city of 11 million persons in the People&rsquo;s Republic of China. Further investigation revealed these cases to result from infection with a newly identified coronavirus, termed the 2019-nCoV. The infection moved rapidly through China, spread to Thailand and Japan, extended into adjacent countries through infected persons travelling by air, eventually reaching multiple countries and continents.", "The infection moved rapidly through China, spread to Thailand and Japan, extended into adjacent countries through infected persons travelling by air, eventually reaching multiple countries and continents. Similar to such other coronaviruses as those causing the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS), the new coronavirus was reported to spread via natural aerosols from human-to-human. In the early stages of this epidemic the case fatality rate is estimated to be approximately 2%, with the majority of deaths occurring in special populations.", "In the early stages of this epidemic the case fatality rate is estimated to be approximately 2%, with the majority of deaths occurring in special populations. Unfortunately, there is limited experience with coronavirus infections during pregnancy, and it now appears certain that pregnant women have become infected during the present 2019-nCoV epidemic. In order to assess the potential of the Wuhan 2019-nCoV to cause maternal, fetal and neonatal morbidity and other poor obstetrical outcomes, this communication reviews the published data addressing the epidemiological and clinical effects of SARS, MERS, and other coronavirus infections on pregnant women and their infants.", "In order to assess the potential of the Wuhan 2019-nCoV to cause maternal, fetal and neonatal morbidity and other poor obstetrical outcomes, this communication reviews the published data addressing the epidemiological and clinical effects of SARS, MERS, and other coronavirus infections on pregnant women and their infants. Recommendations are also made for the consideration of pregnant women in the design, clinical trials, and implementation of future 2019-nCoV vaccines. Text: Coronaviruses are spherical, enveloped, and the largest of positive-strand RNA viruses.", "Text: Coronaviruses are spherical, enveloped, and the largest of positive-strand RNA viruses. They have a wide host range, including birds, farm animals, pets, camels, and bats, in which they primarily cause respiratory and gastrointestinal disease. Belonging to the order Nidovirales, family Coronaviridae, and the subfamily Orthocoronaviridae there are four genera of coronaviruses-Alphacoronavirus, Betacoronavirus, Deltacorona virus, and Gammacoronavirus [1] [2] [3] [4] .", "Belonging to the order Nidovirales, family Coronaviridae, and the subfamily Orthocoronaviridae there are four genera of coronaviruses-Alphacoronavirus, Betacoronavirus, Deltacorona virus, and Gammacoronavirus [1] [2] [3] [4] . In humans, they are a cause of mild illnesses including the common colds occurring in children and adults, and were believed to be of modest medical importance. However, two zoonotic coronaviruses-including the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV)-can produce severe lower respiratory In the beginning of December 2019, a cluster of persons with a pneumonia of unknown cause was identified in Wuhan, the capital of Hubei Province and a large city of approximately 11 million persons located in the central region of the People's Republic of China [7, 8] .", "However, two zoonotic coronaviruses-including the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV)-can produce severe lower respiratory In the beginning of December 2019, a cluster of persons with a pneumonia of unknown cause was identified in Wuhan, the capital of Hubei Province and a large city of approximately 11 million persons located in the central region of the People's Republic of China [7, 8] . Between 8 and 18 December 2019 there were 7 cases of pneumonia identified whose clinical features resembled that of a viral pneumonia. The outbreak was initially believed to be linked to the Wuhan Huanan (South China) Seafood Wholesale Market.", "The outbreak was initially believed to be linked to the Wuhan Huanan (South China) Seafood Wholesale Market. This market, termed a \"wet\" market, sells a variety of seafood, cuts of meat, and both live and dead animals in over one thousand stalls in constant close contact; however, whether this market was the origin of the outbreak remains unknown [9] . On 31 December 2019, the Chinese Center for Disease Control and Prevention (China CDC) sent a rapid response team to Hubei to work alongside health personnel from the provincial and Wuhan city health departments to conduct an epidemiologic investigation.", "On 31 December 2019, the Chinese Center for Disease Control and Prevention (China CDC) sent a rapid response team to Hubei to work alongside health personnel from the provincial and Wuhan city health departments to conduct an epidemiologic investigation. As the disease was spreading through secondary and tertiary cases, the World Health Organization (WHO) China Country Office was informed on 31 December 2019 of the occurrence of these cases of pneumonia of unknown etiology. During the period from 31 December 2019 to 3 January 2020, 44 patients with pneumonia of unknown etiology were reported by the Chinese authorities to the WHO.", "During the period from 31 December 2019 to 3 January 2020, 44 patients with pneumonia of unknown etiology were reported by the Chinese authorities to the WHO. On 7 January 2020 investigators in China identified the etiological agent of the epidemic as a previously unknown coronavirus, and it was given the designation 2019-nCoV (for 2019 novel coronavirus) [8] . Analysis of the clinical features of 41 hospitalized patients with laboratory-confirmed 2019-nCoV infection revealed that 30 were men (73%); less than one-half had underlying co-morbid conditions (13; 32%) which included diabetes (8, 20%) , hypertension (6, 15%), and cardiovascular disease (6; 15%); and the average age was 49.0 years old.", "Analysis of the clinical features of 41 hospitalized patients with laboratory-confirmed 2019-nCoV infection revealed that 30 were men (73%); less than one-half had underlying co-morbid conditions (13; 32%) which included diabetes (8, 20%) , hypertension (6, 15%), and cardiovascular disease (6; 15%); and the average age was 49.0 years old. The most common symptoms at the beginning of their illness included fever (40, 98%) , cough (31, 76%) , and fatigue or myalgia (18, 44%) , sputum production (11, 28%) , and headache (3, 8%) [10] . Among these 41 initial cases of 2019-nCoV infection there were 12 patients (32%) who developed acute respiratory distress syndrome (ARDS), 13 (32%) required intensive care and 6 (15%) died.", "Among these 41 initial cases of 2019-nCoV infection there were 12 patients (32%) who developed acute respiratory distress syndrome (ARDS), 13 (32%) required intensive care and 6 (15%) died. During the first weeks of January the infection spread rapidly through China and extended to adjacent countries where cases began to appear-13 January in Thailand, 15 January in Japan, 20 January in the Republic of Korea, and Taiwan and the United States on 21 January [11] . Infected travelers, mostly via commercial air travel, are known to have been responsible for introducing the virus outside of Wuhan.", "Infected travelers, mostly via commercial air travel, are known to have been responsible for introducing the virus outside of Wuhan. The new coronavirus continued to spread throughout multiple countries and continents, and by 9 February 2020 the WHO reported 37,251 confirmed cases in China that resulted in 812 deaths, surpassing the number of deaths that occurred during the 2002-2003 SARS epidemic. An additional 307 cases of 2019-nCoV infection have occurred among 24 other countries outside of China [12] .", "An additional 307 cases of 2019-nCoV infection have occurred among 24 other countries outside of China [12] . (Figure 1 ) At the meeting of the Emergency Committee of the WHO on 30 January, the novel coronavirus 2019 epidemic was declared a Public Health Emergency of International Concern (PHEIC) [11, 13] . Viruses 2020, 12, 194 3 of 16 epidemic.", "Viruses 2020, 12, 194 3 of 16 epidemic. An additional 307 cases of 2019-nCoV infection have occurred among 24 other countries outside of China [12] . (Figure 1 ) At the meeting of the Emergency Committee of the WHO on 30 January, the novel coronavirus 2019 epidemic was declared a Public Health Emergency of International Concern (PHEIC) [11, 13] .", "(Figure 1 ) At the meeting of the Emergency Committee of the WHO on 30 January, the novel coronavirus 2019 epidemic was declared a Public Health Emergency of International Concern (PHEIC) [11, 13] . This newly recognized coronavirus, producing a disease that has been termed COVID-19, is rapidly spreading throughout China, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV.", "It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV. In order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy. Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women.", "Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. It is the most prevalent non-obstetric infectious condition that occurs during pregnancy [14] [15] [16] . In one study pneumonia was the 3rd most common cause of indirect maternal death [17] .", "In one study pneumonia was the 3rd most common cause of indirect maternal death [17] . Approximately 25 percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [16] . Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] .", "Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] . As with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [19] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [20] [21] [22] . This has been evident historically during previous epidemics.", "This has been evident historically during previous epidemics. The case fatality rate (CFR) for pregnant women infected with influenza during the 1918-1919 pandemic was 27%-even higher when exposure occurred during the 3rd trimester and upwards of 50% if pneumonia supervened [23] . During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] .", "During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] . The most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include This newly recognized coronavirus, producing a disease that has been termed COVID-19, is rapidly spreading throughout China, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV.", "It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV. In order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy. Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women.", "Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. It is the most prevalent non-obstetric infectious condition that occurs during pregnancy [14] [15] [16] . In one study pneumonia was the 3rd most common cause of indirect maternal death [17] .", "In one study pneumonia was the 3rd most common cause of indirect maternal death [17] . Approximately 25 percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [16] . Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] .", "Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] . As with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [19] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [20] [21] [22] . This has been evident historically during previous epidemics.", "This has been evident historically during previous epidemics. The case fatality rate (CFR) for pregnant women infected with influenza during the 1918-1919 pandemic was 27%-even higher when exposure occurred during the 3rd trimester and upwards of 50% if pneumonia supervened [23] . During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] .", "During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] . The most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include premature rupture of membranes (PROM) and preterm labor (PTL), intrauterine fetal demise (IUFD), intrauterine growth restriction (IUGR), and neonatal death [14] [15] [16] . The SARS epidemic began quietly at the turn of the 21st century.", "The SARS epidemic began quietly at the turn of the 21st century. In November 2002, a cook in Guangdong Province, China, died from an unidentified illness. He had worked at a restaurant in which meat from wild animals was served.", "He had worked at a restaurant in which meat from wild animals was served. On 27 November 2002 Chinese-language media and internet reports were picked up by Canada's Global Public Health Intelligence Network (GPHIN) that indicated a flu-like illness was occurring in China [25, 26] . Unfortunately, the reports were not translated, and China failed to report the occurrence of this illness to the World Health Organization (WHO) until February 2003.", "Unfortunately, the reports were not translated, and China failed to report the occurrence of this illness to the World Health Organization (WHO) until February 2003. The disease spread to other countries where it primarily infected healthcare workers. One of these was Dr. Carlo Urbani, a WHO physician investigating a patient with the new disease in Hanoi.", "One of these was Dr. Carlo Urbani, a WHO physician investigating a patient with the new disease in Hanoi. He recognized that the pneumonia was probably caused by a new, highly infectious agent, and rapidly notified the WHO. He contracted the SARS-CoV while there, became febrile and later died after traveling to Thailand to attend a conference.", "He contracted the SARS-CoV while there, became febrile and later died after traveling to Thailand to attend a conference. On 12 March 2003, WHO issued a global alert regarding the disease that was occurring primarily among health care workers in Hanoi, Vietnam and Hong Kong. The disease continued to spread, and by 31 July 2003 there were 8422 probable cases, leading to 916 deaths in 29 countries, with the majority of cases occurring in mainland China and Hong Kong.", "The disease continued to spread, and by 31 July 2003 there were 8422 probable cases, leading to 916 deaths in 29 countries, with the majority of cases occurring in mainland China and Hong Kong. Approximately 30% of infections occurred in healthcare workers. By the termination of the epidemic the global CFR was 11% [27] .", "By the termination of the epidemic the global CFR was 11% [27] . Although there were relatively few documented cases of SARS occurring during pregnancy, several case reports and small clinical studies have described the clinical effects in pregnant women and their infants. In reviewing these reports describing pregnant women with SARS in China it is possible, and perhaps even probable, that some of the same patients were included in more than one publication.", "In reviewing these reports describing pregnant women with SARS in China it is possible, and perhaps even probable, that some of the same patients were included in more than one publication. However, even if this is the case, there is no doubt that SARS coronavirus infection was found to be associated with severe maternal illness, maternal death, and spontaneous abortion [19, [28] [29] [30] [31] . Martha Anker, an expert in statistics formerly with the WHO and the University of Massachusetts, estimated that more than 100 cases of SARS-CoV infection occurred in pregnant women, which warrants closer inspection [27] .", "Martha Anker, an expert in statistics formerly with the WHO and the University of Massachusetts, estimated that more than 100 cases of SARS-CoV infection occurred in pregnant women, which warrants closer inspection [27] . The clinical outcomes among pregnant women with SARS in Hong Kong were worse than those occurring in infected women who were not pregnant [32] . Wong et al.", "Wong et al. [29] evaluated the obstetrical outcomes from a cohort of pregnant women who developed SARS in Hong Kong during the period of 1 February to 31 July 2003. Four of the 7 women (57%) that presented during the 1st trimester sustained spontaneous miscarriages, likely a result of the hypoxia that was caused by SARS-related acute respiratory distress.", "Four of the 7 women (57%) that presented during the 1st trimester sustained spontaneous miscarriages, likely a result of the hypoxia that was caused by SARS-related acute respiratory distress. Among the 5 women who presented after 24 weeks gestation, 4 had preterm deliveries (80%). A case-control study to determine the effects of SARS on pregnancy compared 10 pregnant and 40 non-pregnant women with the infection at the Princess Margaret Hospital in Hong Kong [27, 33] .", "A case-control study to determine the effects of SARS on pregnancy compared 10 pregnant and 40 non-pregnant women with the infection at the Princess Margaret Hospital in Hong Kong [27, 33] . There were 3 deaths among the pregnant women with SARS (maternal mortality rate of 30%) and no deaths in the non-pregnant group of infected women (P = 0.006). Renal failure (P = 0.006) and disseminated intravascular coagulopathy (P = 0.006) developed more frequently in pregnant SARS patients when compared with the non-pregnant SARS group.", "Renal failure (P = 0.006) and disseminated intravascular coagulopathy (P = 0.006) developed more frequently in pregnant SARS patients when compared with the non-pregnant SARS group. Six pregnant women with SARS required admission to the intensive care unit (ICU) (60%) and 4 required endotracheal intubation (40%), compared with a 12.5% intubation rate (P = 0.065) and 17.5% ICU admission rate (P = 0.012) in the non-pregnant group. Maxwell et al.", "Maxwell et al. [32] reported 7 pregnant women infected with SARS-CoV who were followed at a designated SARS unit-2 of the 7 died (CFR of 28%), and 4 (57%) required ICU hospitalization and mechanical ventilation. In contrast, the mortality rate was less than 10% and mechanical ventilation rate less than 20% among non-pregnant, age-matched counterparts who were not infected with SARS-CoV.", "In contrast, the mortality rate was less than 10% and mechanical ventilation rate less than 20% among non-pregnant, age-matched counterparts who were not infected with SARS-CoV. Two women with SARS recovered and maintained their pregnancy but had infants with IUGR. Among the live newborn infants, none had clinical or laboratory evidence for SARS-CoV infection.", "Among the live newborn infants, none had clinical or laboratory evidence for SARS-CoV infection. The new mothers who had developed SARS were advised not to breastfeed to prevent possible vertical transmission of the virus. Zhang et al. [34] described SARS-CoV infections in 5 primagravidas from Guangzhou, China at the height of the SARS epidemic.", "[34] described SARS-CoV infections in 5 primagravidas from Guangzhou, China at the height of the SARS epidemic. Two of the mothers became infected in the 2nd trimester, and 3 developed infection in the 3rd trimester. Two of the pregnant women had hospital-acquired SARS infections, and the other 3 were community-acquired.", "Two of the pregnant women had hospital-acquired SARS infections, and the other 3 were community-acquired. All 5 pregnant women had fever and abnormal chest radiographs; 4 had cough; 4 developed hypoalbuminemia; 3 had elevated alanine aminotransferase levels (ALT), 3 had chills or rigor, 2 had decreased lymphocytes, and 2 had decreased platelets. One pregnant woman required intensive care, but all recovered and there were no maternal deaths.", "One pregnant woman required intensive care, but all recovered and there were no maternal deaths. The 5 infants were clinically evaluated, and none had evidence of SARS. Two pregnant women with SARS were reported from the United States. In a detailed case report, Robertson et al.", "In a detailed case report, Robertson et al. [35] described a 36-year-old pregnant woman with an intermittent cough of approximately 10 days duration and no fever. While travelling in Hong Kong during the 2003 epidemic, she was exposed at her hotel to a person subsequently known to be infected with SARS-CoV.", "While travelling in Hong Kong during the 2003 epidemic, she was exposed at her hotel to a person subsequently known to be infected with SARS-CoV. At 19 weeks gestation she developed fever, anorexia, headache, increasing cough, weakness, and shortness of breath. Upon returning to the United States she was hospitalized with pneumonia.", "Upon returning to the United States she was hospitalized with pneumonia. Obstetrical ultrasounds revealed a low-lying placenta (placenta previa) but were otherwise normal. Following her discharge home and clinical recovery, she was found to have antibodies to SARS-CoV.", "Following her discharge home and clinical recovery, she was found to have antibodies to SARS-CoV. She underwent cesarean section at 38 weeks gestation because of the placenta previa and a healthy baby girl was delivered [35, 36] . The placenta was interpreted as being normal.", "The placenta was interpreted as being normal. At 130 days post-maternal illness, maternal serum and whole blood, swabs from maternal nasopharynx and rectum, post-delivery placenta, umbilical cord blood, amniotic fluid, and breast milk were collected for analysis-no viral RNA was detected in specimens tested by reverse transcriptase polymerase chain reaction (RT-PCR). Antibodies to SARS-CoV were detected from maternal serum, umbilical cord blood, and breast milk by enzyme immunoassay (EIA) and indirect immunofluorescence assay.", "Antibodies to SARS-CoV were detected from maternal serum, umbilical cord blood, and breast milk by enzyme immunoassay (EIA) and indirect immunofluorescence assay. No clinical specimens (except for cord blood) were available for testing from the infant. The second case in the USA occurred in a 38-year-old woman who had travelled to Hong Kong at 7 weeks gestation where she was exposed to SARS-CoV in the same hotel as the aforementioned American woman [37] .", "The second case in the USA occurred in a 38-year-old woman who had travelled to Hong Kong at 7 weeks gestation where she was exposed to SARS-CoV in the same hotel as the aforementioned American woman [37] . Following her return to the United States, her husband developed the clinical onset of SARS, and 6 days later she became ill with fever, myalgia, chills, headache, coryza, and a productive cough with shortness of breath and wheezing. Following her hospitalization for SARS she recovered, serum samples taken on days 28 and 64 post-onset of illness were positive for antibodies to SARS-CoV by enzyme immunoassay and immunofluorescent assays.", "Following her hospitalization for SARS she recovered, serum samples taken on days 28 and 64 post-onset of illness were positive for antibodies to SARS-CoV by enzyme immunoassay and immunofluorescent assays. Her pregnancy continued and was unremarkable except for developing elevated glucose levels. A cesarean section that was performed at 36 weeks gestation due to preterm rupture of membranes and fetal distress resulted in a healthy baby boy.", "A cesarean section that was performed at 36 weeks gestation due to preterm rupture of membranes and fetal distress resulted in a healthy baby boy. At the time of delivery, the mother's serum samples were positive for antibodies to SARS-CoV, but samples taken of umbilical cord blood and placenta were negative. Breast milk sampled 12 and 30 days after delivery were also negative for SARS-CoV antibodies.", "Breast milk sampled 12 and 30 days after delivery were also negative for SARS-CoV antibodies. Specimens evaluated from maternal blood, stool, and nasopharynx samples, as well as umbilical cord blood of the infant, were all negative for coronavirus RNA by RT-PCR. Neonatal stool samples obtained on days-of-life 12 and 30 were also negative for viral RNA.", "Neonatal stool samples obtained on days-of-life 12 and 30 were also negative for viral RNA. From Canada, Yudin et al. [38] reported a 33-year-old pregnant woman who was admitted to the hospital at 31 weeks gestation with a fever, dry cough, and abnormal chest radiograph demonstrating patchy infiltrates.", "[38] reported a 33-year-old pregnant woman who was admitted to the hospital at 31 weeks gestation with a fever, dry cough, and abnormal chest radiograph demonstrating patchy infiltrates. She had acquired SARS from contact with an infected family member. Following a 21-day stay in the hospital, during which she did not require ventilatory support, her convalescent antibody titers were positive for coronavirus infection.", "Following a 21-day stay in the hospital, during which she did not require ventilatory support, her convalescent antibody titers were positive for coronavirus infection. She had a normal labor and delivery and her newborn girl had no evidence of infection. In a study of 5 liveborn neonates who were delivered to women infected with SARS-CoV during the Hong Kong epidemic, results from multiple tests-including serial RT-PCR assays, viral culture, and paired neonatal serological titers-were negative for SARS-CoV [39] .", "In a study of 5 liveborn neonates who were delivered to women infected with SARS-CoV during the Hong Kong epidemic, results from multiple tests-including serial RT-PCR assays, viral culture, and paired neonatal serological titers-were negative for SARS-CoV [39] . None of the 5 neonates developed any clinical signs or symptoms of respiratory infection or compromise. Fortunately, there were no cases of vertical transmission identified among pregnant women infected with SARS-CoV during the 2002-2003 Asian epidemic [27, 30, 31, 39, 40] , and with the exception of a small cluster of cases that recurred in late 2003, no new cases of SARS have occurred.", "Fortunately, there were no cases of vertical transmission identified among pregnant women infected with SARS-CoV during the 2002-2003 Asian epidemic [27, 30, 31, 39, 40] , and with the exception of a small cluster of cases that recurred in late 2003, no new cases of SARS have occurred. In the only reported study of the placental pathology of mothers with SARS, Ng et al. [41] reported the findings from 7 pregnant women infected with SARS-CoV.", "[41] reported the findings from 7 pregnant women infected with SARS-CoV. In the case of 2 women who were convalescing from SARS-CoV infection during the 1st trimester of pregnancy, the placentas were found to be normal. Three placentas were delivered from pregnancies in which the mothers had acute SARS-CoV infection-these were abnormal and demonstrated increased subchorionic and intervillous fibrin, a finding that can be associated with abnormal maternal blood flow to the placenta.", "Three placentas were delivered from pregnancies in which the mothers had acute SARS-CoV infection-these were abnormal and demonstrated increased subchorionic and intervillous fibrin, a finding that can be associated with abnormal maternal blood flow to the placenta. In the placentas of 2 women who were convalescing from SARS-CoV infection in the 3rd trimester of pregnancy the placentas were highly abnormal. They showed extensive fetal thrombotic vasculopathy with areas of avascular chorionic villi-chronic findings of fetal vascular malperfusion.", "They showed extensive fetal thrombotic vasculopathy with areas of avascular chorionic villi-chronic findings of fetal vascular malperfusion. These 2 pregnancies also were complicated by oligohydramnios and had poor obstetrical outcomes-both infants had developed IUGR. It is interesting that villitis, the microscopic finding of inflammation of the chorionic villi that is the histologic hallmark of many maternal hematogenous infections that are transmitted through the placenta to the fetus, was not identified in any of these placentas.", "It is interesting that villitis, the microscopic finding of inflammation of the chorionic villi that is the histologic hallmark of many maternal hematogenous infections that are transmitted through the placenta to the fetus, was not identified in any of these placentas. Similar to other coronavirus infections, SARS-CoV is easily spread from person-to-person via respiratory droplets and secretions as well as through nosocomial contacts [42, 43] . In addition to transmission of SARS-CoV through natural aerosols from infected patients, it was found that in Hong Kong the SARS-CoV could also be transmitted by mechanical aerosols [44] .", "In addition to transmission of SARS-CoV through natural aerosols from infected patients, it was found that in Hong Kong the SARS-CoV could also be transmitted by mechanical aerosols [44] . Environmental factors had an important role when it was discovered that during the Amoy Gardens housing estate outbreak as many as two-thirds of infected persons had diarrhea, SARS-CoV was excreted in their stools, and that aerosols arising from the flushing of toilets could transmit the virus [44] . Healthcare facilities were also an important source of new SARS infections during the 2002-2003 epidemic, and healthcare workers were also at high risk for acquiring the infection.", "Healthcare facilities were also an important source of new SARS infections during the 2002-2003 epidemic, and healthcare workers were also at high risk for acquiring the infection. In order to address the safety issues for the obstetrical management and delivery of pregnant women with SARS, guidelines were prepared by the Canadian Task Force on Preventive Health Care and the Society of Obstetricians and Gynaecologists of Canada [45] . These recommendations include:\n\n1.", "These recommendations include:\n\n1. \"All hospitals should have infection control systems in place to ensure that alerts regarding changes in exposure risk factors for SARS or other potentially serious communicable diseases are conveyed promptly to clinical units, including the labour and delivery unit. At times of SARS outbreaks, all pregnant patients being assessed or admitted to the hospital should be screened for symptoms of and risk factors for SARS.", "At times of SARS outbreaks, all pregnant patients being assessed or admitted to the hospital should be screened for symptoms of and risk factors for SARS. Upon arrival in the labour triage unit, pregnant patients with suspected and probable SARS should be placed in a negative pressure isolation room with at least 6 air exchanges per hour. All labour and delivery units caring for suspected and probable SARS should have available at least one room in which patients can safely labour and deliver while in need of airborne isolation.", "All labour and delivery units caring for suspected and probable SARS should have available at least one room in which patients can safely labour and deliver while in need of airborne isolation. If possible, labour and delivery (including operative delivery or Caesarean section) should be managed in a designated negative pressure isolation room, by designated personnel with specialized infection control preparation and protective gear. 5.", "5. Either regional or general anaesthesia may be appropriate for delivery of patients with SARS. Neonates of mothers with SARS should be isolated in a designated unit until the infant has been well for 10 days, or until the mother's period of isolation is complete. The mother should not breastfeed during this period. 7.", "The mother should not breastfeed during this period. 7. A multidisciplinary team, consisting of obstetricians, nurses, pediatricians, infection control specialists, respiratory therapists, and anaesthesiologists, should be identified in each unit and be responsible for the unit organization and implementation of SARS management protocols. 8.", "8. Staff caring for pregnant SARS patients should not care for other pregnant patients. Staff caring for pregnant SARS patients should be actively monitored for fever and other symptoms of SARS. Such individuals should not work in the presence of any SARS symptoms within 10 days of exposure to a SARS patient. 9.", "9. All health care personnel, trainees, and support staff should be trained in infection control management and containment to prevent spread of the SARS virus. 10.", "10. Regional health authorities in conjunction with hospital staff should consider designating specific facilities or health care units, including primary, secondary, or tertiary health care centers, to care for patients with SARS or similar illnesses.\" Middle East respiratory syndrome (MERS) was first reported in September 2012 in Saudi Arabia, following isolation of MERS-CoV from a male patient who died months earlier from severe pneumonia and multiple organ failure [1] .", "Middle East respiratory syndrome (MERS) was first reported in September 2012 in Saudi Arabia, following isolation of MERS-CoV from a male patient who died months earlier from severe pneumonia and multiple organ failure [1] . In the 8 years since then, there have been more than 2494 confirmed cases of MERS resulting in upwards of 858 deaths globally [46] . While 27 countries have reported cases of MERS, approximately 80% of confirmed cases originated in Saudi Arabia [47] .", "While 27 countries have reported cases of MERS, approximately 80% of confirmed cases originated in Saudi Arabia [47] . To date, all known cases of MERS can be linked to travel or residence in countries along the Arabian Peninsula-that is, Bahrain; Iraq; Iran; Israel, the West Bank, and Gaza; Jordan; Kuwait; Lebanon; Oman; Qatar, Saudi Arabia; Syria; the United Arab Emirates (UAE); and Yemen [48] . The largest documented outbreak outside of this region occurred in 2015 in the Republic of Korea, in which 186 infections occurred, resulting in 38 deaths [49] .", "The largest documented outbreak outside of this region occurred in 2015 in the Republic of Korea, in which 186 infections occurred, resulting in 38 deaths [49] . The index case in this outbreak reportedly returned from the Arabian Peninsula just prior to onset of illness [50] . MERS-CoV is characterized by sporadic zoonotic transmission events as well as spread between infected patients and close contacts (i.e., intra-familial transmission) [51] .", "MERS-CoV is characterized by sporadic zoonotic transmission events as well as spread between infected patients and close contacts (i.e., intra-familial transmission) [51] . Nosocomial outbreaks in health care settings-the result of poor infection control and prevention-are widely recognized as the hallmark of MERS [1] . Superspreading events have been recorded in healthcare settings in Jordan, Al Hasa, Jeddah, Abu Dhabi and South Korea [47, [52] [53] [54] [55] .", "Superspreading events have been recorded in healthcare settings in Jordan, Al Hasa, Jeddah, Abu Dhabi and South Korea [47, [52] [53] [54] [55] . Like other coronaviruses, MERS-CoV can be spread through person-to-person contact, likely via infected respiratory secretions [48] . Transmission dynamics, however, are otherwise poorly understood [1] .", "Transmission dynamics, however, are otherwise poorly understood [1] . Bats are believed to be the natural reservoir of MERS-CoV, and dromedary camels can have the virus and have been suggested as possible intermediary hosts as well as a source of infection to humans [2, 56, 57] . There are no clinical or serological reports of perinatal transmission of MERS, though vertical transmission has been reported for non-coronavirus respiratory viruses including influenza and respiratory syncytial virus (RSV) [58] .", "There are no clinical or serological reports of perinatal transmission of MERS, though vertical transmission has been reported for non-coronavirus respiratory viruses including influenza and respiratory syncytial virus (RSV) [58] . Researchers have not yet discovered ongoing transmission of MERS-CoV within communities outside of health care settings. The clinical presentation of MERS varies from asymptomatic to severe pneumonia with acute respiratory distress syndrome (ARDS), septic shock, and multiple organ failure, often resulting in death.", "The clinical presentation of MERS varies from asymptomatic to severe pneumonia with acute respiratory distress syndrome (ARDS), septic shock, and multiple organ failure, often resulting in death. Most patients with MERS develop severe acute respiratory illness accompanied by fever, cough, and shortness of breath [50] . Progression to pneumonia is swift-usually within the first week -and at least one-third of patients also present with gastrointestinal symptoms [1] .", "Progression to pneumonia is swift-usually within the first week -and at least one-third of patients also present with gastrointestinal symptoms [1] . MERS progresses much more rapidly to respiratory failure and has a higher case fatality rate than SARS [1] . Unlike SARS, however, infection with MERS-CoV is generally mild in healthy individuals but more severe in immunocompromised patients and people with underlying comorbidities [1] .", "Unlike SARS, however, infection with MERS-CoV is generally mild in healthy individuals but more severe in immunocompromised patients and people with underlying comorbidities [1] . The overall CFR of MERS is approximately 34.4% [46] . Most fatalities have been associated with pre-existing medical conditions like chronic lung disease, diabetes, and renal failure, as well as weakened immune systems [59] , making such individuals high risk.", "Most fatalities have been associated with pre-existing medical conditions like chronic lung disease, diabetes, and renal failure, as well as weakened immune systems [59] , making such individuals high risk. As a result of the immunological changes that occur during pregnancy, women who are pregnant are included in this high-risk group. Pregnant women may develop severe disease and fatal maternal and/or fetal outcomes as a result of MERS-CoV infection; however, little is known of the pathophysiology of this infection during pregnancy.", "Pregnant women may develop severe disease and fatal maternal and/or fetal outcomes as a result of MERS-CoV infection; however, little is known of the pathophysiology of this infection during pregnancy. Limited data exists on the prevalence and clinical features of MERS during pregnancy, birth, and the postnatal period. It is likely, however, that the immunological changes that normally occur in pregnancy may alter susceptibility to the MERS-CoV and the severity of clinical illness [60] .", "It is likely, however, that the immunological changes that normally occur in pregnancy may alter susceptibility to the MERS-CoV and the severity of clinical illness [60] . Pregnant women infected with SARS-CoV, a related coronavirus, appear to have increased morbidity and mortality when compared to non-pregnant women, suggesting that MERS-CoV could also lead to severe clinical outcomes in pregnancy. To date, however, very few pregnancy-associated cases (n = 11) have been documented, with 91% having adverse clinical outcomes.", "To date, however, very few pregnancy-associated cases (n = 11) have been documented, with 91% having adverse clinical outcomes. Between November 2012 and February 2016, there were 1308 cases of MERS reported by the Saudi Arabia Ministry of Health (MoH). Of these, 5 patients were pregnant, according to a retrospective study by Assiri et al.", "Of these, 5 patients were pregnant, according to a retrospective study by Assiri et al. [47] , and all resulted in adverse outcomes. Patient ages ranged from 27 to 34 years, with occurrence of exposure in either the 2nd or 3rd trimester. All 5 cases received intensive care.", "All 5 cases received intensive care. Two women died and there were 2 cases of perinatal death-1 stillbirth and 1 neonatal death shortly after emergency cesarean section. These instances of severe maternal and perinatal outcomes are consistent with other reports of MERS-CoV infection in pregnant women, as well as outcomes associated with SARS-CoV infection.", "These instances of severe maternal and perinatal outcomes are consistent with other reports of MERS-CoV infection in pregnant women, as well as outcomes associated with SARS-CoV infection. The authors of the retrospectives study concede that unreported cases of MERS in pregnancy are likely due to lack of routine pregnancy testing [47] . They conclude that pregnancy testing for women of reproductive age should be considered for those who test positive for MERS-CoV, to contribute to overall understanding of pathogenesis and epidemiological risk.", "They conclude that pregnancy testing for women of reproductive age should be considered for those who test positive for MERS-CoV, to contribute to overall understanding of pathogenesis and epidemiological risk. Additionally, 2 of the 5 patients were healthcare workers, which corresponds with existing knowledge of higher risk of exposure to MERS-CoV in healthcare settings. In a separate case report of MERS occurring in pregnancy, Alserehi et al.", "In a separate case report of MERS occurring in pregnancy, Alserehi et al. [58] described a 33-year-old critical care nurse who became infected during the 3rd trimester in the midst of a large hospital outbreak. In the days following hospital admission, she developed respiratory failure necessitating mechanical ventilation and administration of dexamethasone as prophylaxis for the fetus.", "In the days following hospital admission, she developed respiratory failure necessitating mechanical ventilation and administration of dexamethasone as prophylaxis for the fetus. Following an emergency cesarean section at 32 weeks gestation, she was transferred to the intensive care unit (ICU) and later recovered. The preterm but otherwise healthy infant was kept in the neonatal unit for observation and later released along with his mother.", "The preterm but otherwise healthy infant was kept in the neonatal unit for observation and later released along with his mother. In contrast to other reported cases, this patient had a successful outcome, perhaps due to the timing of MERS-CoV exposure, her young age, the use of steroids, and differences in immune response. Alfaraj et al.", "Alfaraj et al. [61] described 2 cases of maternal infection with MERS-CoV at the Prince Mohammed Bin Abdulaziz Hospital (PMAH) in Saudi Arabia. Maternal infection in both cases was confirmed by nasopharyngeal swab testing by RT-PCR. One patient was a 29-year-old woman at 6 weeks gestation with no underlying medical conditions.", "One patient was a 29-year-old woman at 6 weeks gestation with no underlying medical conditions. The second patient, a 39-year-old at 24 weeks gestation, had several comorbidities, including end stage renal disease, hypertension, and hemodialysis. This woman presented to the hospital after contact with a MERS-CoV-infected person during an active outbreak.", "This woman presented to the hospital after contact with a MERS-CoV-infected person during an active outbreak. Both patients later tested negative for MERS-CoV and were subsequently discharged. The younger patient delivered a healthy, full-term infant. The status of the other delivery is unknown. Neither fetus was tested for MERS-CoV. According to Payne et al.", "Neither fetus was tested for MERS-CoV. According to Payne et al. [62] , epidemiologic investigation of the 2012 MERS outbreak in Zarqa, Jordan, revealed that a 2nd trimester stillbirth (5 months gestational age) had occurred as a result of maternal exposure to MERS-CoV.", "[62] , epidemiologic investigation of the 2012 MERS outbreak in Zarqa, Jordan, revealed that a 2nd trimester stillbirth (5 months gestational age) had occurred as a result of maternal exposure to MERS-CoV. The mother experienced fever, fatigue, headache and cough, concurrently with vaginal bleeding and abdominal pain. On the 7th day of symptoms, she had a fetal death.", "On the 7th day of symptoms, she had a fetal death. The mother was confirmed to have antibody to MERS-CoV, and she self-reported having had unprotected contact with family members who later tested positive for the virus. This was the first documented occurrence of stillbirth during maternal infection with MERS-CoV.", "This was the first documented occurrence of stillbirth during maternal infection with MERS-CoV. On 24 November 2013, a 32-year-old pregnant woman in the United Arab Emirates (UAE) developed ARDS following admission to the ICU after suspected community-acquired pneumonia advanced to respiratory failure and hypotension [60] . Later that day, her baby was delivered by caesarean section and subsequent Apgar scores were within healthy range.", "Later that day, her baby was delivered by caesarean section and subsequent Apgar scores were within healthy range. The next day, RT-PCR evaluation revealed that the mother was positive for MERS-CoV. Despite rigorous intervention, including oral ribavirin-peginterferon-α therapy and ventilator support, the woman continued to deteriorate, developed septic shock, and died.", "Despite rigorous intervention, including oral ribavirin-peginterferon-α therapy and ventilator support, the woman continued to deteriorate, developed septic shock, and died. While the outcome for this mother was fatal, Malik et al. noted that virus shedding ceased during therapy with ribavirin and peginterferon-α and radiographic evidence indicated clinical improvement before her death [58] .", "noted that virus shedding ceased during therapy with ribavirin and peginterferon-α and radiographic evidence indicated clinical improvement before her death [58] . More research is needed to determine safety, efficacy, and dosage of these therapies in the general population but also in pregnant women. While few data exist on the effects of these treatments in pregnant humans, ribavirin is generally contraindicated during pregnancy [58] .", "While few data exist on the effects of these treatments in pregnant humans, ribavirin is generally contraindicated during pregnancy [58] . Outside of the Middle East the only confirmed case of MERS in pregnancy occurred in 2015 in South Korea. Jeong et al.", "Jeong et al. [49] reported that a 39-year-old patient was exposed during the 3rd trimester following contact with a patient having MERS. Despite abrupt vaginal bleeding and rupture of membranes, the patient recovered fully and delivered a healthy infant at 37 weeks and 5 days gestation.", "Despite abrupt vaginal bleeding and rupture of membranes, the patient recovered fully and delivered a healthy infant at 37 weeks and 5 days gestation. Subsequent testing of the infant's blood did not detect any IgG, IgM, or IgA antibodies to MERS-CoV. The mean maternal age of the 11 confirmed maternal SARS cases described above was 33.2 years, with a mean gestational age of 26.3 weeks.", "The mean maternal age of the 11 confirmed maternal SARS cases described above was 33.2 years, with a mean gestational age of 26.3 weeks. The source of infection in 2 of the cases was attributed to contact with family members who tested positive for MERS-CoV, unknown in 3 cases, likely due to animal exposure in 1 case, and 6 were healthcare-associated (2 of these patients were healthcare workers). Six patients required intensive care and 3 died.", "Six patients required intensive care and 3 died. Of those who died, 2 were exposed to MERS-CoV in the 3rd trimester, and 1 was exposed during the 2nd trimester. The infant death rate for all 11 cases was 27%.", "The infant death rate for all 11 cases was 27%. Fetal survival did not appear to correlate with the timing of maternal infection and gestational age; however, more data are needed to draw conclusions about this relationship. According to Alfaraj et al.", "According to Alfaraj et al. [61] , the CFR for the 11 infected women-also 27%-was not statistically different from the overall CFR of MERS in the general population (35%) (P = 0.75). Only 1 case resulted in both maternal and fetal death.", "Only 1 case resulted in both maternal and fetal death. Similar to SARS in pregnancy, more research is needed to understand the pathogenesis and epidemiology of MERS in pregnancy including the relationship between the timing of maternal infection, gestational age of the fetus, the effects of comorbid factors, and the occurrence of adverse outcomes. Few studies documented the presence of MERS-CoV antibodies in the umbilical cord or neonatal blood, making it difficult to assess perinatal transmission.", "Few studies documented the presence of MERS-CoV antibodies in the umbilical cord or neonatal blood, making it difficult to assess perinatal transmission. As such, future studies should involve the collection of samples from relevant specimens including amniotic fluid, placenta, and umbilical cord [49] . MERS prevention should be high priority for high-risk exposures such as healthcare workers, pregnant women and individuals working with camels, camel meat-milk processors and in abattoirs [57] .", "MERS prevention should be high priority for high-risk exposures such as healthcare workers, pregnant women and individuals working with camels, camel meat-milk processors and in abattoirs [57] . Since 2013, the Saudi Arabia MoH has recommended that pregnant women postpone travel to Saudi Arabia for the Hajj and Umrah [47] . To further reduce risk of exposure among pregnant women, additional measures such as avoiding contact with camels and sick persons-particularly in healthcare settings-are also recommended.", "To further reduce risk of exposure among pregnant women, additional measures such as avoiding contact with camels and sick persons-particularly in healthcare settings-are also recommended. Pregnant women who present with symptoms of pneumonia, influenza-like illness (ILI), or sepsis on the Arabian Peninsula may also benefit from MERS-CoV screening to expedite early diagnosis and improve disease management [60] . While multiple agents have been used to treat MERS, none have been tested in large clinical studies.", "While multiple agents have been used to treat MERS, none have been tested in large clinical studies. Available data are limited to the use of combination therapies of interferon and other agents in case reports and case series [63] . A prospective or randomized study may prove difficult given the sporadic nature of MERS-CoV outbreaks.", "A prospective or randomized study may prove difficult given the sporadic nature of MERS-CoV outbreaks. Due to a gap in research on the treatment of MERS in pregnancy, there are no therapeutic options currently recommended for pregnant women [58] . Therapies under development and testing may be considered inappropriate for pregnant women due to the unknown potential for teratogenic effects.", "Therapies under development and testing may be considered inappropriate for pregnant women due to the unknown potential for teratogenic effects. For example, during the 2003 SARS outbreak, ribavirin was administered to pregnant women with severe cases of the disease, but ribavirin therapy has been documented to increase the risk of teratogenic effects in newborns [58] . The Alphacoronaviruses HCoV 229E and NL63, as well as the Betacoronaviruses HKU 1 and OC43, can infect humans and cause the common cold.", "The Alphacoronaviruses HCoV 229E and NL63, as well as the Betacoronaviruses HKU 1 and OC43, can infect humans and cause the common cold. In order to investigate the potential maternal-fetal transmission of human coronaviruses during pregnancy, Gagneur et al. [64, 65] evaluated 3 types of maternal-infant paired specimens that included maternal vaginal and respiratory specimens that were obtained during labor, as well as gastric samples from the newborn infants.", "[64, 65] evaluated 3 types of maternal-infant paired specimens that included maternal vaginal and respiratory specimens that were obtained during labor, as well as gastric samples from the newborn infants. These specimens were evaluated for the presence of HCoV 229E, OC-43, NL63 and HKU 1 using RT-PCR methodology. Between the period from July 2003 to August 2005 the authors examined 159 mother-infant dyads.", "Between the period from July 2003 to August 2005 the authors examined 159 mother-infant dyads. Human coronaviruses were identified in 12 samples (HCoV 229E: 11; HKU 1 : 1) from 7 mother-child pairs. In 3 mother-infant dyads only maternal respiratory samples were positive; in 2 other pairs all 3 of the samples tested positive for human coronavirus; in 1 case only the maternal vaginal and newborn gastric samples were positive; and in another case the maternal vaginal sample alone was positive.", "In 3 mother-infant dyads only maternal respiratory samples were positive; in 2 other pairs all 3 of the samples tested positive for human coronavirus; in 1 case only the maternal vaginal and newborn gastric samples were positive; and in another case the maternal vaginal sample alone was positive. There were no signs of clinical infection in any of the 3 neonates that had positive gastric samples for human coronavirus. It is beyond the scope of this communication to discuss the various technical challenges inherent in developing a safe and efficacious vaccine for coronavirus infections in humans.", "It is beyond the scope of this communication to discuss the various technical challenges inherent in developing a safe and efficacious vaccine for coronavirus infections in humans. There are clearly challenges to this endeavor-protective antibodies to coronaviruses are not long-lasting, tissue damage has been reported to occur as a result of exposure to SARS-CoV, development of animal models that closely resemble human infection are limited, and the extensive time and expense necessary to perform clinical trials in humans, to name a few [66] [67] [68] . It is vitally important that pregnant women be considered in the design, clinical trial, and implementation of vaccine candidates for 2019-nCoV.", "It is vitally important that pregnant women be considered in the design, clinical trial, and implementation of vaccine candidates for 2019-nCoV. In examining the history of vaccine design, it is clear that the needs of pregnant women have rarely been prioritized in either the preclinical development or the clinical trial phases of production. Today, pregnant women are usually excluded from experimental trial of drugs and vaccines that do not target obstetric conditions [69] .", "Today, pregnant women are usually excluded from experimental trial of drugs and vaccines that do not target obstetric conditions [69] . Excluding pregnant women and their infants from participation in vaccine development and implementation undermines ethical principles of justice-fairness, equity, and maximization of benefit-and potentially places their health at risk during outbreaks and other health emergencies [69] [70] [71] . On 23 January 2020 the Coalition for Epidemic Preparedness Innovations (CEPI) announced three programs to develop a vaccine against the novel Wuhan coronavirus.", "On 23 January 2020 the Coalition for Epidemic Preparedness Innovations (CEPI) announced three programs to develop a vaccine against the novel Wuhan coronavirus. The Chief Executive Officer of CEPI, Richard Hatchett, said [72] :\n\n\"Given the rapid global spread of the nCoV-2019 virus the world needs to act quickly and in unity to tackle this disease. Our intention with this work is to leverage our work on the MERS coronavirus and rapid response platforms to speed up vaccine development.\"", "Our intention with this work is to leverage our work on the MERS coronavirus and rapid response platforms to speed up vaccine development.\" The novel coronavirus is the first epidemic disease to emerge since the formation of CEPI in Davos in 2017. CEPI was created with the express intent to enable speedy research and development of vaccines against emerging pathogens.", "CEPI was created with the express intent to enable speedy research and development of vaccines against emerging pathogens. In May 2017, WHO released the Target Product Profile (TPP) for MERS-CoV vaccines, following the prioritization of MERS-CoV as one of eight priority pathogens for prevention of epidemics [73] . CEPI and partners aim to use existing platforms-that is, the existing \"backbone\" that can be adapted for use against new pathogens-that are currently in preclinical development for MERS-CoV vaccine candidates.", "CEPI and partners aim to use existing platforms-that is, the existing \"backbone\" that can be adapted for use against new pathogens-that are currently in preclinical development for MERS-CoV vaccine candidates. Following the WHO declaration on 30 January that the current 2019-nCoV outbreak is a public health emergency of international concern (PHEIC), global health organizations and researchers will be further mobilized-bolstered by new mechanisms for action and greater resources-to stop the spread of disease. A critical question that must be answered at this stage-with a clear view of the potential deleterious effects of a new coronavirus in pregnancy-is will maternal immunization be a priority in research and development?", "A critical question that must be answered at this stage-with a clear view of the potential deleterious effects of a new coronavirus in pregnancy-is will maternal immunization be a priority in research and development? As of the PHEIC declaration, 12 groups have announced that they are developing new vaccines against 2019-nCoV and seven others announced initiatives to develop new therapies [74] . Safe testing of experimental vaccines in a pregnant population is difficult and, as a result, vaccines are not typically developed with pregnant women in mind.", "Safe testing of experimental vaccines in a pregnant population is difficult and, as a result, vaccines are not typically developed with pregnant women in mind. To date, very few clinical trials for vaccines have proactively included pregnant women [75] , and the exclusion of pregnant and lactating women from receiving the rVSV-ZEBOV vaccine through 3 Ebola virus epidemics serves as a recent example [69] [70] [71] . Given the potential severity in pregnancy, as demonstrated by this review of maternal infections of SARS and MERS, women who are pregnant should be considered a priority population in all efforts to prepare for and prevent infection by novel coronaviruses.", "Given the potential severity in pregnancy, as demonstrated by this review of maternal infections of SARS and MERS, women who are pregnant should be considered a priority population in all efforts to prepare for and prevent infection by novel coronaviruses. On 5 February 2020 it was reported by multiple media outlets that a newborn infant delivered during the epidemic in Wuhan had tested positive for 2019-nCoV at the Wuhan Children's Hospital in Hubei Province 30 hours following its birth. According to the official Xinhua news agency, the infant was delivered on 2 February to a mother who had tested positive for the virus.", "According to the official Xinhua news agency, the infant was delivered on 2 February to a mother who had tested positive for the virus. Reports have stated that the infant had stable vital signs, no fever or cough, but had shortness of breath together with abnormal chest radiographs and abnormalities of liver function [76] [77] [78] . Dr. Zeng Lingkong, Chief Physician at the Neonatal Medicine Department of the hospital, said [78] , \"This reminds us to pay attention to mother-to-child being a possible route of coronavirus transmission\"\n\nThe hospital also provided information about a previous case of a baby that had been delivered on 13 January 2020.", "Dr. Zeng Lingkong, Chief Physician at the Neonatal Medicine Department of the hospital, said [78] , \"This reminds us to pay attention to mother-to-child being a possible route of coronavirus transmission\"\n\nThe hospital also provided information about a previous case of a baby that had been delivered on 13 January 2020. Following its birth, the infant's nanny was diagnosed with 2019-nCoV, and the mother was diagnosed days later [76] . On 29 January the baby began to develop symptoms.", "On 29 January the baby began to develop symptoms. According to Dr. Zeng Lingkong [76] , \"Whether it was the baby's nanny who passed the virus to the mother who passed it to the baby, we cannot be sure at the moment. But we can confirm that the baby was in close contact with patients infected with the new coronavirus, which says newborns can also be infected\"\n\nIn considering whether these and future cases of neonatal infection are acquired prior to delivery, it is important to remember that newborn infants can acquire an infection in other ways beyond intrauterine maternal-fetal transmission.", "But we can confirm that the baby was in close contact with patients infected with the new coronavirus, which says newborns can also be infected\"\n\nIn considering whether these and future cases of neonatal infection are acquired prior to delivery, it is important to remember that newborn infants can acquire an infection in other ways beyond intrauterine maternal-fetal transmission. In some cases, viral infection can be acquired when the infant passes through the birth canal during a vaginal delivery or through post-partum breast feeding, although these mechanisms would be highly unusual for a respiratory virus. Neonatal infection from respiratory viruses can occur after delivery through such mechanisms as inhalation of the agent through aerosols produced by coughing from the mother, relatives or healthcare workers or other sources in the hospital environment.", "Neonatal infection from respiratory viruses can occur after delivery through such mechanisms as inhalation of the agent through aerosols produced by coughing from the mother, relatives or healthcare workers or other sources in the hospital environment. Based upon past experience with pregnant women who developed MERS and SARS, and realizing that the numbers are limited, there has never been confirmed intrauterine coronavirus transmission from mother to fetus. Discussing the most recent baby to be diagnosed with the 2019-nCoV infection, Dr. Stephen Morse, an epidemiologist at the Mailman School of Public Health at Columbia University stated [77] , \"It's more likely that the baby contracted the virus from the hospital environment, the same way healthcare workers get infected by the patients they treat,\" \"It's quite possible that the baby picked it up very conventionally-by inhaling virus droplets that came from the mother coughing.\"", "Discussing the most recent baby to be diagnosed with the 2019-nCoV infection, Dr. Stephen Morse, an epidemiologist at the Mailman School of Public Health at Columbia University stated [77] , \"It's more likely that the baby contracted the virus from the hospital environment, the same way healthcare workers get infected by the patients they treat,\" \"It's quite possible that the baby picked it up very conventionally-by inhaling virus droplets that came from the mother coughing.\" And according to Dr. Paul Hunter, Professor of Medicine at the University of East Anglia [79] , \"As far as I am aware there is currently no evidence that the novel coronavirus can be transmitted in the womb. When a baby is born vaginally it is exposed to the mother's gut microbiome, therefore if a baby does get infected with coronavirus a few days after birth we currently cannot tell if the baby was infected in the womb or during birth.\"", "When a baby is born vaginally it is exposed to the mother's gut microbiome, therefore if a baby does get infected with coronavirus a few days after birth we currently cannot tell if the baby was infected in the womb or during birth.\" There is limited knowledge regarding coronavirus infections that occur during pregnancy-what is known has, for the most part, been the result of epidemics resulting from two different diseases, SARS and MERS. These previous experiences with coronavirus infections in pregnancy indicates that these agents are capable of causing adverse clinical outcomes including life-threatening maternal disease that in some cases requires hospitalization, intensive care and ventilatory support.", "These previous experiences with coronavirus infections in pregnancy indicates that these agents are capable of causing adverse clinical outcomes including life-threatening maternal disease that in some cases requires hospitalization, intensive care and ventilatory support. Both of these coronaviruses can result in maternal death in a small but significant number of cases, but the specific risk factors for a fatal outcome during pregnancy have not been clarified. Coronaviruses can also result in adverse outcomes for the fetus and infant including intrauterine growth restriction, preterm delivery, admission to the ICU, spontaneous abortion and perinatal death.", "Coronaviruses can also result in adverse outcomes for the fetus and infant including intrauterine growth restriction, preterm delivery, admission to the ICU, spontaneous abortion and perinatal death. Unlike some viral infections, notably Ebola virus [70] and Zika virus [80] , the likelihood of intrauterine maternal-fetal transmission of coronaviruses is low-there have been no documented cases of vertical transmission occurring with either SARS or MERS. It remains to be seen during the current Wuhan 2019-nCoV epidemic how this newly-emergent coronavirus affects pregnant women and their infants, as well as which factors may modulate obstetrical disease and outcomes including the timing of maternal coronavirus exposure by gestational age, the effects of medications or other treatment regimens, differences in host immune responses, occurrence of coexisting medical and obstetrical conditions, and other covariables.", "It remains to be seen during the current Wuhan 2019-nCoV epidemic how this newly-emergent coronavirus affects pregnant women and their infants, as well as which factors may modulate obstetrical disease and outcomes including the timing of maternal coronavirus exposure by gestational age, the effects of medications or other treatment regimens, differences in host immune responses, occurrence of coexisting medical and obstetrical conditions, and other covariables. However, pregnant women should be considered to be at high risk for developing severe infection during this current outbreak of 2019-nCoV. Additional clinical research on the treatment of SARS, MERS, and the new coronavirus 2019-nCoV is necessary if we are to understand the potential risks and benefits of novel therapies and new vaccines in pregnancy.", "Additional clinical research on the treatment of SARS, MERS, and the new coronavirus 2019-nCoV is necessary if we are to understand the potential risks and benefits of novel therapies and new vaccines in pregnancy. This research will be critical in improving the care, and even saving the lives, of pregnant women in the current as well as future outbreaks." ]

[ "To understand the time-dependent risk of infection on a cruise ship, the Diamond Princess, I estimated the incidence of infection with novel coronavirus (COVID-19). The epidemic curve of a total of 199 confirmed cases was drawn, classifying individuals into passengers with and without close contact and crew members. A backcalculation method was employed to estimate the incidence of infection.", "A backcalculation method was employed to estimate the incidence of infection. The peak time of infection was seen for the time period from 2 to 4 February 2020, and the incidence has abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed.", "The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed. Without the intervention from 5 February, it was predicted that the cumulative incidence with and without close contact would have been as large as 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively, while these were kept to be 102 and 47 cases, respectively. Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited.", "Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited. Movement restriction greatly reduced the number of infections from 5 February onwards. Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] .", "Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] . The primary case remains unknown, but the index case, defined as the first identified case, is a passenger who started coughing from 19 January 2020 on board, disembarking the ship in Hong Kong on 25 January. As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards.", "As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards. Subsequently, the movement of all passengers was restricted on board from 5 February, for a matter of 14 days of quarantine. Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported.", "Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported. One of the critical issues in infectious disease epidemiology is that the time of infection event is seldom directly observable. For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] .", "For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] . Using a sophisticated statistical model with doubly intervalcensored likelihood and right truncation with an exponential growth of cases, the mean incubation period has been estimated to be about 5.0 days [3] . To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure.", "To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure. I analyzed the epidemic curve, ct, on day t, illustrated by the number of confirmed cases by the date of illness onset. The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR).", "The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR). The date of illness onset was defined as the first date of fever. In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger.", "In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger. Close contact was defined as having at least one cabinmate who was confirmed by RT-PCR. We estimate the number of cases by time of infection, it.", "We estimate the number of cases by time of infection, it. Using the probability mass function of the incubation period of length s, fs, the incidence of infection is known to satisfy\n\nwhere E(.) represents the expected value.", "represents the expected value. As for fs, it is known that the mean and standard deviation are 5.0 and 3.0 days, respectively, best fitted by lognormal distribution [3] . Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method.", "Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method. The estimation was implemented independently by the history of contact and type of membership. Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve.", "Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve. Assuming that the cumulative incidence is Gaussian distributed, four unknown parameters were estimated. The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020.", "The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020. The latter dataset corresponds to the time period without any impact of movement restriction that was in place from 5 February onwards. Figure 1 shows the epidemic curve by contact history and type of membership.", "Figure 1 shows the epidemic curve by contact history and type of membership. The highest incidence of illness onset was observed on 7 February. The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship.", "The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship. The second dominating group was passengers with close contact history. The last illness onset date on board of a passenger without close contact was on 14 February.", "The last illness onset date on board of a passenger without close contact was on 14 February. Estimating the incidence of infection, the peak incidence was identified for the period from 2 to 4 February among passengers both with and without close contact (Figure 2 ). The incidence of infection abruptly dropped after 5 February, the date of movement restriction.", "The incidence of infection abruptly dropped after 5 February, the date of movement restriction. Among passengers without close contact, the incidence was estimated to be zero, except for 8-10 February 2020, during which 0.98 persons (95% confidence intervals (CI): 0, 7.74) per day were estimated to have been infected. The epidemic peak among crew members was seen for the period from 8 to 10 February 2020.", "The epidemic peak among crew members was seen for the period from 8 to 10 February 2020. Figure 3 compares the cumulative incidence with and without movement restriction policy from 5 February. In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020.", "In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020. These were well realized by the Richards model. Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively.", "Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively. A large outbreak of COVID-19 occurred on a cruise ship. Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards.", "Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February, on which the movement restriction policy was imposed, and at most there was, on average, one case of infection per day from 8 to 10 February. Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board.", "Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board. Based on an analysis of illness onset data on board (and before the disembarkation of a large number of passengers), the risk of infection among passengers without close contact was considered to be very limited Among disembarked passengers, symptomatic cases have started to be reported on the ground in and outside of Japan. In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases.", "In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases. Although the transmission via direct human-to-human contact was prevented by movement restrictions, the role of other modes of transmission, e.g., environmental and asymptomatic transmissions, should be further explored. The author declares no conflict of interest." ]

[ "To understand the time-dependent risk of infection on a cruise ship, the Diamond Princess, I estimated the incidence of infection with novel coronavirus (COVID-19). The epidemic curve of a total of 199 confirmed cases was drawn, classifying individuals into passengers with and without close contact and crew members. A backcalculation method was employed to estimate the incidence of infection.", "A backcalculation method was employed to estimate the incidence of infection. The peak time of infection was seen for the time period from 2 to 4 February 2020, and the incidence has abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed.", "The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed. Without the intervention from 5 February, it was predicted that the cumulative incidence with and without close contact would have been as large as 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively, while these were kept to be 102 and 47 cases, respectively. Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited.", "Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited. Movement restriction greatly reduced the number of infections from 5 February onwards. Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] .", "Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] . The primary case remains unknown, but the index case, defined as the first identified case, is a passenger who started coughing from 19 January 2020 on board, disembarking the ship in Hong Kong on 25 January. As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards.", "As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards. Subsequently, the movement of all passengers was restricted on board from 5 February, for a matter of 14 days of quarantine. Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported.", "Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported. One of the critical issues in infectious disease epidemiology is that the time of infection event is seldom directly observable. For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] .", "For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] . Using a sophisticated statistical model with doubly intervalcensored likelihood and right truncation with an exponential growth of cases, the mean incubation period has been estimated to be about 5.0 days [3] . To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure.", "To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure. I analyzed the epidemic curve, ct, on day t, illustrated by the number of confirmed cases by the date of illness onset. The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR).", "The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR). The date of illness onset was defined as the first date of fever. In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger.", "In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger. Close contact was defined as having at least one cabinmate who was confirmed by RT-PCR. We estimate the number of cases by time of infection, it.", "We estimate the number of cases by time of infection, it. Using the probability mass function of the incubation period of length s, fs, the incidence of infection is known to satisfy\n\nwhere E(.) represents the expected value.", "represents the expected value. As for fs, it is known that the mean and standard deviation are 5.0 and 3.0 days, respectively, best fitted by lognormal distribution [3] . Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method.", "Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method. The estimation was implemented independently by the history of contact and type of membership. Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve.", "Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve. Assuming that the cumulative incidence is Gaussian distributed, four unknown parameters were estimated. The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020.", "The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020. The latter dataset corresponds to the time period without any impact of movement restriction that was in place from 5 February onwards. Figure 1 shows the epidemic curve by contact history and type of membership.", "Figure 1 shows the epidemic curve by contact history and type of membership. The highest incidence of illness onset was observed on 7 February. The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship.", "The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship. The second dominating group was passengers with close contact history. The last illness onset date on board of a passenger without close contact was on 14 February.", "The last illness onset date on board of a passenger without close contact was on 14 February. Estimating the incidence of infection, the peak incidence was identified for the period from 2 to 4 February among passengers both with and without close contact (Figure 2 ). The incidence of infection abruptly dropped after 5 February, the date of movement restriction.", "The incidence of infection abruptly dropped after 5 February, the date of movement restriction. Among passengers without close contact, the incidence was estimated to be zero, except for 8-10 February 2020, during which 0.98 persons (95% confidence intervals (CI): 0, 7.74) per day were estimated to have been infected. The epidemic peak among crew members was seen for the period from 8 to 10 February 2020.", "The epidemic peak among crew members was seen for the period from 8 to 10 February 2020. Figure 3 compares the cumulative incidence with and without movement restriction policy from 5 February. In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020.", "In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020. These were well realized by the Richards model. Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively.", "Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively. A large outbreak of COVID-19 occurred on a cruise ship. Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards.", "Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February, on which the movement restriction policy was imposed, and at most there was, on average, one case of infection per day from 8 to 10 February. Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board.", "Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board. Based on an analysis of illness onset data on board (and before the disembarkation of a large number of passengers), the risk of infection among passengers without close contact was considered to be very limited Among disembarked passengers, symptomatic cases have started to be reported on the ground in and outside of Japan. In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases.", "In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases. Although the transmission via direct human-to-human contact was prevented by movement restrictions, the role of other modes of transmission, e.g., environmental and asymptomatic transmissions, should be further explored. The author declares no conflict of interest." ]

[ "To understand the time-dependent risk of infection on a cruise ship, the Diamond Princess, I estimated the incidence of infection with novel coronavirus (COVID-19). The epidemic curve of a total of 199 confirmed cases was drawn, classifying individuals into passengers with and without close contact and crew members. A backcalculation method was employed to estimate the incidence of infection.", "A backcalculation method was employed to estimate the incidence of infection. The peak time of infection was seen for the time period from 2 to 4 February 2020, and the incidence has abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed.", "The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed. Without the intervention from 5 February, it was predicted that the cumulative incidence with and without close contact would have been as large as 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively, while these were kept to be 102 and 47 cases, respectively. Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited.", "Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited. Movement restriction greatly reduced the number of infections from 5 February onwards. Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] .", "Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] . The primary case remains unknown, but the index case, defined as the first identified case, is a passenger who started coughing from 19 January 2020 on board, disembarking the ship in Hong Kong on 25 January. As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards.", "As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards. Subsequently, the movement of all passengers was restricted on board from 5 February, for a matter of 14 days of quarantine. Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported.", "Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported. One of the critical issues in infectious disease epidemiology is that the time of infection event is seldom directly observable. For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] .", "For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] . Using a sophisticated statistical model with doubly intervalcensored likelihood and right truncation with an exponential growth of cases, the mean incubation period has been estimated to be about 5.0 days [3] . To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure.", "To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure. I analyzed the epidemic curve, ct, on day t, illustrated by the number of confirmed cases by the date of illness onset. The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR).", "The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR). The date of illness onset was defined as the first date of fever. In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger.", "In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger. Close contact was defined as having at least one cabinmate who was confirmed by RT-PCR. We estimate the number of cases by time of infection, it.", "We estimate the number of cases by time of infection, it. Using the probability mass function of the incubation period of length s, fs, the incidence of infection is known to satisfy\n\nwhere E(.) represents the expected value.", "represents the expected value. As for fs, it is known that the mean and standard deviation are 5.0 and 3.0 days, respectively, best fitted by lognormal distribution [3] . Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method.", "Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method. The estimation was implemented independently by the history of contact and type of membership. Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve.", "Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve. Assuming that the cumulative incidence is Gaussian distributed, four unknown parameters were estimated. The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020.", "The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020. The latter dataset corresponds to the time period without any impact of movement restriction that was in place from 5 February onwards. Figure 1 shows the epidemic curve by contact history and type of membership.", "Figure 1 shows the epidemic curve by contact history and type of membership. The highest incidence of illness onset was observed on 7 February. The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship.", "The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship. The second dominating group was passengers with close contact history. The last illness onset date on board of a passenger without close contact was on 14 February.", "The last illness onset date on board of a passenger without close contact was on 14 February. Estimating the incidence of infection, the peak incidence was identified for the period from 2 to 4 February among passengers both with and without close contact (Figure 2 ). The incidence of infection abruptly dropped after 5 February, the date of movement restriction.", "The incidence of infection abruptly dropped after 5 February, the date of movement restriction. Among passengers without close contact, the incidence was estimated to be zero, except for 8-10 February 2020, during which 0.98 persons (95% confidence intervals (CI): 0, 7.74) per day were estimated to have been infected. The epidemic peak among crew members was seen for the period from 8 to 10 February 2020.", "The epidemic peak among crew members was seen for the period from 8 to 10 February 2020. Figure 3 compares the cumulative incidence with and without movement restriction policy from 5 February. In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020.", "In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020. These were well realized by the Richards model. Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively.", "Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively. A large outbreak of COVID-19 occurred on a cruise ship. Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards.", "Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February, on which the movement restriction policy was imposed, and at most there was, on average, one case of infection per day from 8 to 10 February. Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board.", "Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board. Based on an analysis of illness onset data on board (and before the disembarkation of a large number of passengers), the risk of infection among passengers without close contact was considered to be very limited Among disembarked passengers, symptomatic cases have started to be reported on the ground in and outside of Japan. In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases.", "In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases. Although the transmission via direct human-to-human contact was prevented by movement restrictions, the role of other modes of transmission, e.g., environmental and asymptomatic transmissions, should be further explored. The author declares no conflict of interest." ]

[ "To understand the time-dependent risk of infection on a cruise ship, the Diamond Princess, I estimated the incidence of infection with novel coronavirus (COVID-19). The epidemic curve of a total of 199 confirmed cases was drawn, classifying individuals into passengers with and without close contact and crew members. A backcalculation method was employed to estimate the incidence of infection.", "A backcalculation method was employed to estimate the incidence of infection. The peak time of infection was seen for the time period from 2 to 4 February 2020, and the incidence has abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed.", "The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed. Without the intervention from 5 February, it was predicted that the cumulative incidence with and without close contact would have been as large as 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively, while these were kept to be 102 and 47 cases, respectively. Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited.", "Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited. Movement restriction greatly reduced the number of infections from 5 February onwards. Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] .", "Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] . The primary case remains unknown, but the index case, defined as the first identified case, is a passenger who started coughing from 19 January 2020 on board, disembarking the ship in Hong Kong on 25 January. As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards.", "As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards. Subsequently, the movement of all passengers was restricted on board from 5 February, for a matter of 14 days of quarantine. Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported.", "Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported. One of the critical issues in infectious disease epidemiology is that the time of infection event is seldom directly observable. For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] .", "For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] . Using a sophisticated statistical model with doubly intervalcensored likelihood and right truncation with an exponential growth of cases, the mean incubation period has been estimated to be about 5.0 days [3] . To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure.", "To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure. I analyzed the epidemic curve, ct, on day t, illustrated by the number of confirmed cases by the date of illness onset. The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR).", "The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR). The date of illness onset was defined as the first date of fever. In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger.", "In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger. Close contact was defined as having at least one cabinmate who was confirmed by RT-PCR. We estimate the number of cases by time of infection, it.", "We estimate the number of cases by time of infection, it. Using the probability mass function of the incubation period of length s, fs, the incidence of infection is known to satisfy\n\nwhere E(.) represents the expected value.", "represents the expected value. As for fs, it is known that the mean and standard deviation are 5.0 and 3.0 days, respectively, best fitted by lognormal distribution [3] . Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method.", "Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method. The estimation was implemented independently by the history of contact and type of membership. Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve.", "Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve. Assuming that the cumulative incidence is Gaussian distributed, four unknown parameters were estimated. The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020.", "The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020. The latter dataset corresponds to the time period without any impact of movement restriction that was in place from 5 February onwards. Figure 1 shows the epidemic curve by contact history and type of membership.", "Figure 1 shows the epidemic curve by contact history and type of membership. The highest incidence of illness onset was observed on 7 February. The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship.", "The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship. The second dominating group was passengers with close contact history. The last illness onset date on board of a passenger without close contact was on 14 February.", "The last illness onset date on board of a passenger without close contact was on 14 February. Estimating the incidence of infection, the peak incidence was identified for the period from 2 to 4 February among passengers both with and without close contact (Figure 2 ). The incidence of infection abruptly dropped after 5 February, the date of movement restriction.", "The incidence of infection abruptly dropped after 5 February, the date of movement restriction. Among passengers without close contact, the incidence was estimated to be zero, except for 8-10 February 2020, during which 0.98 persons (95% confidence intervals (CI): 0, 7.74) per day were estimated to have been infected. The epidemic peak among crew members was seen for the period from 8 to 10 February 2020.", "The epidemic peak among crew members was seen for the period from 8 to 10 February 2020. Figure 3 compares the cumulative incidence with and without movement restriction policy from 5 February. In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020.", "In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020. These were well realized by the Richards model. Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively.", "Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively. A large outbreak of COVID-19 occurred on a cruise ship. Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards.", "Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February, on which the movement restriction policy was imposed, and at most there was, on average, one case of infection per day from 8 to 10 February. Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board.", "Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board. Based on an analysis of illness onset data on board (and before the disembarkation of a large number of passengers), the risk of infection among passengers without close contact was considered to be very limited Among disembarked passengers, symptomatic cases have started to be reported on the ground in and outside of Japan. In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases.", "In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases. Although the transmission via direct human-to-human contact was prevented by movement restrictions, the role of other modes of transmission, e.g., environmental and asymptomatic transmissions, should be further explored. The author declares no conflict of interest." ]

[ "To understand the time-dependent risk of infection on a cruise ship, the Diamond Princess, I estimated the incidence of infection with novel coronavirus (COVID-19). The epidemic curve of a total of 199 confirmed cases was drawn, classifying individuals into passengers with and without close contact and crew members. A backcalculation method was employed to estimate the incidence of infection.", "A backcalculation method was employed to estimate the incidence of infection. The peak time of infection was seen for the time period from 2 to 4 February 2020, and the incidence has abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed.", "The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed. Without the intervention from 5 February, it was predicted that the cumulative incidence with and without close contact would have been as large as 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively, while these were kept to be 102 and 47 cases, respectively. Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited.", "Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited. Movement restriction greatly reduced the number of infections from 5 February onwards. Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] .", "Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] . The primary case remains unknown, but the index case, defined as the first identified case, is a passenger who started coughing from 19 January 2020 on board, disembarking the ship in Hong Kong on 25 January. As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards.", "As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards. Subsequently, the movement of all passengers was restricted on board from 5 February, for a matter of 14 days of quarantine. Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported.", "Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported. One of the critical issues in infectious disease epidemiology is that the time of infection event is seldom directly observable. For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] .", "For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] . Using a sophisticated statistical model with doubly intervalcensored likelihood and right truncation with an exponential growth of cases, the mean incubation period has been estimated to be about 5.0 days [3] . To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure.", "To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure. I analyzed the epidemic curve, ct, on day t, illustrated by the number of confirmed cases by the date of illness onset. The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR).", "The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR). The date of illness onset was defined as the first date of fever. In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger.", "In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger. Close contact was defined as having at least one cabinmate who was confirmed by RT-PCR. We estimate the number of cases by time of infection, it.", "We estimate the number of cases by time of infection, it. Using the probability mass function of the incubation period of length s, fs, the incidence of infection is known to satisfy\n\nwhere E(.) represents the expected value.", "represents the expected value. As for fs, it is known that the mean and standard deviation are 5.0 and 3.0 days, respectively, best fitted by lognormal distribution [3] . Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method.", "Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method. The estimation was implemented independently by the history of contact and type of membership. Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve.", "Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve. Assuming that the cumulative incidence is Gaussian distributed, four unknown parameters were estimated. The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020.", "The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020. The latter dataset corresponds to the time period without any impact of movement restriction that was in place from 5 February onwards. Figure 1 shows the epidemic curve by contact history and type of membership.", "Figure 1 shows the epidemic curve by contact history and type of membership. The highest incidence of illness onset was observed on 7 February. The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship.", "The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship. The second dominating group was passengers with close contact history. The last illness onset date on board of a passenger without close contact was on 14 February.", "The last illness onset date on board of a passenger without close contact was on 14 February. Estimating the incidence of infection, the peak incidence was identified for the period from 2 to 4 February among passengers both with and without close contact (Figure 2 ). The incidence of infection abruptly dropped after 5 February, the date of movement restriction.", "The incidence of infection abruptly dropped after 5 February, the date of movement restriction. Among passengers without close contact, the incidence was estimated to be zero, except for 8-10 February 2020, during which 0.98 persons (95% confidence intervals (CI): 0, 7.74) per day were estimated to have been infected. The epidemic peak among crew members was seen for the period from 8 to 10 February 2020.", "The epidemic peak among crew members was seen for the period from 8 to 10 February 2020. Figure 3 compares the cumulative incidence with and without movement restriction policy from 5 February. In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020.", "In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020. These were well realized by the Richards model. Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively.", "Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively. A large outbreak of COVID-19 occurred on a cruise ship. Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards.", "Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February, on which the movement restriction policy was imposed, and at most there was, on average, one case of infection per day from 8 to 10 February. Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board.", "Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board. Based on an analysis of illness onset data on board (and before the disembarkation of a large number of passengers), the risk of infection among passengers without close contact was considered to be very limited Among disembarked passengers, symptomatic cases have started to be reported on the ground in and outside of Japan. In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases.", "In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases. Although the transmission via direct human-to-human contact was prevented by movement restrictions, the role of other modes of transmission, e.g., environmental and asymptomatic transmissions, should be further explored. The author declares no conflict of interest." ]

[ "To understand the time-dependent risk of infection on a cruise ship, the Diamond Princess, I estimated the incidence of infection with novel coronavirus (COVID-19). The epidemic curve of a total of 199 confirmed cases was drawn, classifying individuals into passengers with and without close contact and crew members. A backcalculation method was employed to estimate the incidence of infection.", "A backcalculation method was employed to estimate the incidence of infection. The peak time of infection was seen for the time period from 2 to 4 February 2020, and the incidence has abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed.", "The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed. Without the intervention from 5 February, it was predicted that the cumulative incidence with and without close contact would have been as large as 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively, while these were kept to be 102 and 47 cases, respectively. Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited.", "Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited. Movement restriction greatly reduced the number of infections from 5 February onwards. Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] .", "Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] . The primary case remains unknown, but the index case, defined as the first identified case, is a passenger who started coughing from 19 January 2020 on board, disembarking the ship in Hong Kong on 25 January. As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards.", "As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards. Subsequently, the movement of all passengers was restricted on board from 5 February, for a matter of 14 days of quarantine. Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported.", "Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported. One of the critical issues in infectious disease epidemiology is that the time of infection event is seldom directly observable. For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] .", "For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] . Using a sophisticated statistical model with doubly intervalcensored likelihood and right truncation with an exponential growth of cases, the mean incubation period has been estimated to be about 5.0 days [3] . To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure.", "To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure. I analyzed the epidemic curve, ct, on day t, illustrated by the number of confirmed cases by the date of illness onset. The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR).", "The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR). The date of illness onset was defined as the first date of fever. In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger.", "In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger. Close contact was defined as having at least one cabinmate who was confirmed by RT-PCR. We estimate the number of cases by time of infection, it.", "We estimate the number of cases by time of infection, it. Using the probability mass function of the incubation period of length s, fs, the incidence of infection is known to satisfy\n\nwhere E(.) represents the expected value.", "represents the expected value. As for fs, it is known that the mean and standard deviation are 5.0 and 3.0 days, respectively, best fitted by lognormal distribution [3] . Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method.", "Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method. The estimation was implemented independently by the history of contact and type of membership. Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve.", "Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve. Assuming that the cumulative incidence is Gaussian distributed, four unknown parameters were estimated. The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020.", "The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020. The latter dataset corresponds to the time period without any impact of movement restriction that was in place from 5 February onwards. Figure 1 shows the epidemic curve by contact history and type of membership.", "Figure 1 shows the epidemic curve by contact history and type of membership. The highest incidence of illness onset was observed on 7 February. The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship.", "The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship. The second dominating group was passengers with close contact history. The last illness onset date on board of a passenger without close contact was on 14 February.", "The last illness onset date on board of a passenger without close contact was on 14 February. Estimating the incidence of infection, the peak incidence was identified for the period from 2 to 4 February among passengers both with and without close contact (Figure 2 ). The incidence of infection abruptly dropped after 5 February, the date of movement restriction.", "The incidence of infection abruptly dropped after 5 February, the date of movement restriction. Among passengers without close contact, the incidence was estimated to be zero, except for 8-10 February 2020, during which 0.98 persons (95% confidence intervals (CI): 0, 7.74) per day were estimated to have been infected. The epidemic peak among crew members was seen for the period from 8 to 10 February 2020.", "The epidemic peak among crew members was seen for the period from 8 to 10 February 2020. Figure 3 compares the cumulative incidence with and without movement restriction policy from 5 February. In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020.", "In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020. These were well realized by the Richards model. Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively.", "Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively. A large outbreak of COVID-19 occurred on a cruise ship. Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards.", "Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February, on which the movement restriction policy was imposed, and at most there was, on average, one case of infection per day from 8 to 10 February. Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board.", "Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board. Based on an analysis of illness onset data on board (and before the disembarkation of a large number of passengers), the risk of infection among passengers without close contact was considered to be very limited Among disembarked passengers, symptomatic cases have started to be reported on the ground in and outside of Japan. In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases.", "In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases. Although the transmission via direct human-to-human contact was prevented by movement restrictions, the role of other modes of transmission, e.g., environmental and asymptomatic transmissions, should be further explored. The author declares no conflict of interest." ]

[ "To understand the time-dependent risk of infection on a cruise ship, the Diamond Princess, I estimated the incidence of infection with novel coronavirus (COVID-19). The epidemic curve of a total of 199 confirmed cases was drawn, classifying individuals into passengers with and without close contact and crew members. A backcalculation method was employed to estimate the incidence of infection.", "A backcalculation method was employed to estimate the incidence of infection. The peak time of infection was seen for the time period from 2 to 4 February 2020, and the incidence has abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed.", "The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed. Without the intervention from 5 February, it was predicted that the cumulative incidence with and without close contact would have been as large as 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively, while these were kept to be 102 and 47 cases, respectively. Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited.", "Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited. Movement restriction greatly reduced the number of infections from 5 February onwards. Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] .", "Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] . The primary case remains unknown, but the index case, defined as the first identified case, is a passenger who started coughing from 19 January 2020 on board, disembarking the ship in Hong Kong on 25 January. As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards.", "As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards. Subsequently, the movement of all passengers was restricted on board from 5 February, for a matter of 14 days of quarantine. Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported.", "Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported. One of the critical issues in infectious disease epidemiology is that the time of infection event is seldom directly observable. For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] .", "For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] . Using a sophisticated statistical model with doubly intervalcensored likelihood and right truncation with an exponential growth of cases, the mean incubation period has been estimated to be about 5.0 days [3] . To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure.", "To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure. I analyzed the epidemic curve, ct, on day t, illustrated by the number of confirmed cases by the date of illness onset. The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR).", "The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR). The date of illness onset was defined as the first date of fever. In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger.", "In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger. Close contact was defined as having at least one cabinmate who was confirmed by RT-PCR. We estimate the number of cases by time of infection, it.", "We estimate the number of cases by time of infection, it. Using the probability mass function of the incubation period of length s, fs, the incidence of infection is known to satisfy\n\nwhere E(.) represents the expected value.", "represents the expected value. As for fs, it is known that the mean and standard deviation are 5.0 and 3.0 days, respectively, best fitted by lognormal distribution [3] . Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method.", "Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method. The estimation was implemented independently by the history of contact and type of membership. Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve.", "Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve. Assuming that the cumulative incidence is Gaussian distributed, four unknown parameters were estimated. The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020.", "The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020. The latter dataset corresponds to the time period without any impact of movement restriction that was in place from 5 February onwards. Figure 1 shows the epidemic curve by contact history and type of membership.", "Figure 1 shows the epidemic curve by contact history and type of membership. The highest incidence of illness onset was observed on 7 February. The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship.", "The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship. The second dominating group was passengers with close contact history. The last illness onset date on board of a passenger without close contact was on 14 February.", "The last illness onset date on board of a passenger without close contact was on 14 February. Estimating the incidence of infection, the peak incidence was identified for the period from 2 to 4 February among passengers both with and without close contact (Figure 2 ). The incidence of infection abruptly dropped after 5 February, the date of movement restriction.", "The incidence of infection abruptly dropped after 5 February, the date of movement restriction. Among passengers without close contact, the incidence was estimated to be zero, except for 8-10 February 2020, during which 0.98 persons (95% confidence intervals (CI): 0, 7.74) per day were estimated to have been infected. The epidemic peak among crew members was seen for the period from 8 to 10 February 2020.", "The epidemic peak among crew members was seen for the period from 8 to 10 February 2020. Figure 3 compares the cumulative incidence with and without movement restriction policy from 5 February. In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020.", "In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020. These were well realized by the Richards model. Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively.", "Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively. A large outbreak of COVID-19 occurred on a cruise ship. Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards.", "Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February, on which the movement restriction policy was imposed, and at most there was, on average, one case of infection per day from 8 to 10 February. Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board.", "Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board. Based on an analysis of illness onset data on board (and before the disembarkation of a large number of passengers), the risk of infection among passengers without close contact was considered to be very limited Among disembarked passengers, symptomatic cases have started to be reported on the ground in and outside of Japan. In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases.", "In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases. Although the transmission via direct human-to-human contact was prevented by movement restrictions, the role of other modes of transmission, e.g., environmental and asymptomatic transmissions, should be further explored. The author declares no conflict of interest." ]

[ "To understand the time-dependent risk of infection on a cruise ship, the Diamond Princess, I estimated the incidence of infection with novel coronavirus (COVID-19). The epidemic curve of a total of 199 confirmed cases was drawn, classifying individuals into passengers with and without close contact and crew members. A backcalculation method was employed to estimate the incidence of infection.", "A backcalculation method was employed to estimate the incidence of infection. The peak time of infection was seen for the time period from 2 to 4 February 2020, and the incidence has abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed.", "The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed. Without the intervention from 5 February, it was predicted that the cumulative incidence with and without close contact would have been as large as 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively, while these were kept to be 102 and 47 cases, respectively. Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited.", "Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited. Movement restriction greatly reduced the number of infections from 5 February onwards. Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] .", "Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] . The primary case remains unknown, but the index case, defined as the first identified case, is a passenger who started coughing from 19 January 2020 on board, disembarking the ship in Hong Kong on 25 January. As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards.", "As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards. Subsequently, the movement of all passengers was restricted on board from 5 February, for a matter of 14 days of quarantine. Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported.", "Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported. One of the critical issues in infectious disease epidemiology is that the time of infection event is seldom directly observable. For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] .", "For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] . Using a sophisticated statistical model with doubly intervalcensored likelihood and right truncation with an exponential growth of cases, the mean incubation period has been estimated to be about 5.0 days [3] . To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure.", "To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure. I analyzed the epidemic curve, ct, on day t, illustrated by the number of confirmed cases by the date of illness onset. The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR).", "The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR). The date of illness onset was defined as the first date of fever. In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger.", "In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger. Close contact was defined as having at least one cabinmate who was confirmed by RT-PCR. We estimate the number of cases by time of infection, it.", "We estimate the number of cases by time of infection, it. Using the probability mass function of the incubation period of length s, fs, the incidence of infection is known to satisfy\n\nwhere E(.) represents the expected value.", "represents the expected value. As for fs, it is known that the mean and standard deviation are 5.0 and 3.0 days, respectively, best fitted by lognormal distribution [3] . Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method.", "Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method. The estimation was implemented independently by the history of contact and type of membership. Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve.", "Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve. Assuming that the cumulative incidence is Gaussian distributed, four unknown parameters were estimated. The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020.", "The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020. The latter dataset corresponds to the time period without any impact of movement restriction that was in place from 5 February onwards. Figure 1 shows the epidemic curve by contact history and type of membership.", "Figure 1 shows the epidemic curve by contact history and type of membership. The highest incidence of illness onset was observed on 7 February. The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship.", "The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship. The second dominating group was passengers with close contact history. The last illness onset date on board of a passenger without close contact was on 14 February.", "The last illness onset date on board of a passenger without close contact was on 14 February. Estimating the incidence of infection, the peak incidence was identified for the period from 2 to 4 February among passengers both with and without close contact (Figure 2 ). The incidence of infection abruptly dropped after 5 February, the date of movement restriction.", "The incidence of infection abruptly dropped after 5 February, the date of movement restriction. Among passengers without close contact, the incidence was estimated to be zero, except for 8-10 February 2020, during which 0.98 persons (95% confidence intervals (CI): 0, 7.74) per day were estimated to have been infected. The epidemic peak among crew members was seen for the period from 8 to 10 February 2020.", "The epidemic peak among crew members was seen for the period from 8 to 10 February 2020. Figure 3 compares the cumulative incidence with and without movement restriction policy from 5 February. In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020.", "In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020. These were well realized by the Richards model. Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively.", "Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively. A large outbreak of COVID-19 occurred on a cruise ship. Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards.", "Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February, on which the movement restriction policy was imposed, and at most there was, on average, one case of infection per day from 8 to 10 February. Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board.", "Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board. Based on an analysis of illness onset data on board (and before the disembarkation of a large number of passengers), the risk of infection among passengers without close contact was considered to be very limited Among disembarked passengers, symptomatic cases have started to be reported on the ground in and outside of Japan. In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases.", "In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases. Although the transmission via direct human-to-human contact was prevented by movement restrictions, the role of other modes of transmission, e.g., environmental and asymptomatic transmissions, should be further explored. The author declares no conflict of interest." ]

[ "To understand the time-dependent risk of infection on a cruise ship, the Diamond Princess, I estimated the incidence of infection with novel coronavirus (COVID-19). The epidemic curve of a total of 199 confirmed cases was drawn, classifying individuals into passengers with and without close contact and crew members. A backcalculation method was employed to estimate the incidence of infection.", "A backcalculation method was employed to estimate the incidence of infection. The peak time of infection was seen for the time period from 2 to 4 February 2020, and the incidence has abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed.", "The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed. Without the intervention from 5 February, it was predicted that the cumulative incidence with and without close contact would have been as large as 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively, while these were kept to be 102 and 47 cases, respectively. Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited.", "Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited. Movement restriction greatly reduced the number of infections from 5 February onwards. Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] .", "Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] . The primary case remains unknown, but the index case, defined as the first identified case, is a passenger who started coughing from 19 January 2020 on board, disembarking the ship in Hong Kong on 25 January. As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards.", "As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards. Subsequently, the movement of all passengers was restricted on board from 5 February, for a matter of 14 days of quarantine. Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported.", "Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported. One of the critical issues in infectious disease epidemiology is that the time of infection event is seldom directly observable. For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] .", "For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] . Using a sophisticated statistical model with doubly intervalcensored likelihood and right truncation with an exponential growth of cases, the mean incubation period has been estimated to be about 5.0 days [3] . To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure.", "To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure. I analyzed the epidemic curve, ct, on day t, illustrated by the number of confirmed cases by the date of illness onset. The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR).", "The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR). The date of illness onset was defined as the first date of fever. In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger.", "In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger. Close contact was defined as having at least one cabinmate who was confirmed by RT-PCR. We estimate the number of cases by time of infection, it.", "We estimate the number of cases by time of infection, it. Using the probability mass function of the incubation period of length s, fs, the incidence of infection is known to satisfy\n\nwhere E(.) represents the expected value.", "represents the expected value. As for fs, it is known that the mean and standard deviation are 5.0 and 3.0 days, respectively, best fitted by lognormal distribution [3] . Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method.", "Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method. The estimation was implemented independently by the history of contact and type of membership. Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve.", "Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] :\n\nwhere is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve. Assuming that the cumulative incidence is Gaussian distributed, four unknown parameters were estimated. The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020.", "The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020. The latter dataset corresponds to the time period without any impact of movement restriction that was in place from 5 February onwards. Figure 1 shows the epidemic curve by contact history and type of membership.", "Figure 1 shows the epidemic curve by contact history and type of membership. The highest incidence of illness onset was observed on 7 February. The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship.", "The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship. The second dominating group was passengers with close contact history. The last illness onset date on board of a passenger without close contact was on 14 February.", "The last illness onset date on board of a passenger without close contact was on 14 February. Estimating the incidence of infection, the peak incidence was identified for the period from 2 to 4 February among passengers both with and without close contact (Figure 2 ). The incidence of infection abruptly dropped after 5 February, the date of movement restriction.", "The incidence of infection abruptly dropped after 5 February, the date of movement restriction. Among passengers without close contact, the incidence was estimated to be zero, except for 8-10 February 2020, during which 0.98 persons (95% confidence intervals (CI): 0, 7.74) per day were estimated to have been infected. The epidemic peak among crew members was seen for the period from 8 to 10 February 2020.", "The epidemic peak among crew members was seen for the period from 8 to 10 February 2020. Figure 3 compares the cumulative incidence with and without movement restriction policy from 5 February. In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020.", "In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020. These were well realized by the Richards model. Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively.", "Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively. A large outbreak of COVID-19 occurred on a cruise ship. Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards.", "Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February, on which the movement restriction policy was imposed, and at most there was, on average, one case of infection per day from 8 to 10 February. Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board.", "Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board. Based on an analysis of illness onset data on board (and before the disembarkation of a large number of passengers), the risk of infection among passengers without close contact was considered to be very limited Among disembarked passengers, symptomatic cases have started to be reported on the ground in and outside of Japan. In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases.", "In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases. Although the transmission via direct human-to-human contact was prevented by movement restrictions, the role of other modes of transmission, e.g., environmental and asymptomatic transmissions, should be further explored. The author declares no conflict of interest." ]

[ "Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2]. The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number.", "Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid- to late-January. Average delays between infection and illness onset have been estimated at around 5–6 days, with an upper limit of around 11-14 days [2,5], and delays from illness onset to laboratory confirmation added a further 10 days on average [2].", "Average delays between infection and illness onset have been estimated at around 5–6 days, with an upper limit of around 11-14 days [2,5], and delays from illness onset to laboratory confirmation added a further 10 days on average [2]. Text: It is now 6 weeks since Chinese health authorities announced the discovery of a novel coronavirus (2019-nCoV) [1] causing a cluster of pneumonia cases in Wuhan, the major transport hub of central China. The earliest human infections had occurred by early December 2019, and a large wet market in central Wuhan was linked to most, but not all, of the initial cases [2] .", "The earliest human infections had occurred by early December 2019, and a large wet market in central Wuhan was linked to most, but not all, of the initial cases [2] . While evidence from the initial outbreak investigations seemed to suggest that 2019-nCoV could not easily spread between humans [3] , it is now very clear that infections have been spreading from person to person [2] . We recently estimated that more than 75,000 infections may have occurred in Wuhan as at 25 January 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland China and around the world.", "We recently estimated that more than 75,000 infections may have occurred in Wuhan as at 25 January 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland China and around the world. A number of important characteristics of 2019-nCoV infection have already been identified, but in order to calibrate public health responses we need improved information on transmission dynamics, severity of the disease, immunity, and the impact of control and mitigation measures that have been applied to date. Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] .", "Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] . The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number.", "Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid-to late-January. Average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] .", "Average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] . Chains of transmission have now been reported in a number of locations outside of mainland China. Within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of Hubei province in China, or in other countries.", "Within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of Hubei province in China, or in other countries. If sustained transmission does occur in other locations, it would be valuable to determine whether there is variation in transmissibility by location, for example because of different behaviours or control measures, or because of different environmental conditions. To address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-nCoV transmission to occur.", "To address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-nCoV transmission to occur. In an analysis of the first 425 confirmed cases of infection, 73% of cases with illness onset between 12 and 22 January reported no exposure to either a wet market or another person with symptoms of a respiratory illness [2] . The lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but China's health minister publicly warned that pre-symptomatic transmission could be occurring [6] .", "The lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but China's health minister publicly warned that pre-symptomatic transmission could be occurring [6] . Determining the extent to which asymptomatic or pre-symptomatic transmission might be occurring is an urgent priority, because it has direct implications for public health and hospital infection control. Data on viral shedding dynamics could help in assessing duration of infectiousness.", "Data on viral shedding dynamics could help in assessing duration of infectiousness. For severe acute respiratory syndrome-related coronavirus (SARS-CoV), infectivity peaked at around 10 days after illness onset [7] , consistent with the peak in viral load at around that time [8] . This allowed control of the SARS epidemic through prompt detection of cases and strict isolation.", "This allowed control of the SARS epidemic through prompt detection of cases and strict isolation. For influenza virus infections, virus shedding is highest on the day of illness onset and relatively higher from shortly before symptom onset until a few days after onset [9] . To date, transmission patterns of 2019-nCoV appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than SARS.", "To date, transmission patterns of 2019-nCoV appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than SARS. Transmission of respiratory viruses generally happens through large respiratory droplets, but some respiratory viruses can spread through fine particle aerosols [10] , and indirect transmission via fomites can also play a role. Coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance.", "Coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance. The SARS-CoV superspreading event at Amoy Gardens where more than 300 cases were infected was attributed to faecal-oral, then airborne, spread through pressure differentials between contaminated effluent pipes, bathroom floor drains and flushing toilets [13] . The first large identifiable superspreading event during the present 2019-nCoV outbreak has apparently taken place on the Diamond Princess cruise liner quarantined off the coast of Yokohama, Japan, with at least 130 passengers tested positive for 2019-nCoV as at 10 February 2020 [14] .", "The first large identifiable superspreading event during the present 2019-nCoV outbreak has apparently taken place on the Diamond Princess cruise liner quarantined off the coast of Yokohama, Japan, with at least 130 passengers tested positive for 2019-nCoV as at 10 February 2020 [14] . Identifying which modes are important for 2019-nCoV transmission would inform the importance of personal protective measures such as face masks (and specifically which types) and hand hygiene. The first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had Modelling studies incorporating healthcare capacity and processes pneumonia.", "The first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had Modelling studies incorporating healthcare capacity and processes pneumonia. It is well established that some infections can be severe, particularly in older adults with underlying medical conditions [15, 16] , but based on the generally mild clinical presentation of 2019-nCoV cases detected outside China, it appears that there could be many more mild infections than severe infections. Determining the spectrum of clinical manifestations of 2019-nCoV infections is perhaps the most urgent research priority, because it determines the strength of public health response required.", "Determining the spectrum of clinical manifestations of 2019-nCoV infections is perhaps the most urgent research priority, because it determines the strength of public health response required. If the seriousness of infection is similar to the 1918/19 Spanish influenza, and therefore at the upper end of severity scales in influenza pandemic plans, the same responses would be warranted for 2019-nCoV as for the most severe influenza pandemics. If, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly.", "If, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly. Beyond a robust assessment of overall severity, it is also important to determine high risk groups. Infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] .", "Infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] . Those under 18 years are a critical group to study in order to tease out the relative roles of susceptibility vs severity as possible underlying causes for the very rare recorded instances of infection in this age group. Are children protected from infection or do they not fall ill after infection?", "Are children protected from infection or do they not fall ill after infection? If they are naturally immune, which is unlikely, we should understand why; otherwise, even if they do not show symptoms, it is important to know if they shed the virus. Obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity.", "Obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity. Answers to these questions are especially pertinent as basis for decisions on school closure as a social distancing intervention, which can be hugely disruptive not only for students but also because of its knock-on effect for child care and parental duties. Very few children have been confirmed 2019-nCoV cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers.", "Very few children have been confirmed 2019-nCoV cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers. Serosurveys in affected locations could inform this, in addition to truly assessing the clinical severity spectrum. Another question on susceptibility is regarding whether 2019-nCoV infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera.", "Another question on susceptibility is regarding whether 2019-nCoV infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera. Some experts already questioned whether the 2019-nCoV may behave similarly to MERS-CoV in cases exhibiting mild symptoms without eliciting neutralising antibodies [17] . A separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] .", "A separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] . If either of these were to be relevant, the transmission dynamics could become more complex. A wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-nCoV.", "A wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-nCoV. Internationally, the past week has seen an increasing number of countries issue travel advisories or outright entry bans on persons from Hubei province or China as a whole, as well as substantial cuts in flights to and from affected areas out of commercial considerations. Evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions.", "Evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions. If and when local transmission begins in a particular location, a variety of community mitigation measures can be implemented by health authorities to reduce transmission and thus reduce the growth rate of an epidemic, reduce the height of the epidemic peak and the peak demand on healthcare services, as well as reduce the total number of infected persons [21] . A number of social distancing measures have already been implemented in Chinese cities in the past few weeks including school and workplace closures.", "A number of social distancing measures have already been implemented in Chinese cities in the past few weeks including school and workplace closures. It should now be an urgent priority to quantify the effects of these measures and specifically whether they can reduce the effective reproductive number below 1, because this will guide the response strategies in other locations. During the 1918/19 influenza pandemic, cities in the United States, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] .", "During the 1918/19 influenza pandemic, cities in the United States, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] . Similarly to international travel interventions, local social distancing measures should be assessed for their impact and when they could be safely discontinued, albeit in a coordinated and deliberate manner across China such that recrudescence in the epidemic curve is minimised. Mobile telephony global positioning system (GPS) data and location services data from social media providers such as Baidu and Tencent in China could become the first occasion when these data inform outbreak control in real time.", "Mobile telephony global positioning system (GPS) data and location services data from social media providers such as Baidu and Tencent in China could become the first occasion when these data inform outbreak control in real time. At the individual level, surgical face masks have often been a particularly visible image from affected cities in China. Face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons.", "Face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons. However, there is now a shortage of supply of masks in China and elsewhere, and debates are ongoing about their protective value for uninfected persons in the general community. The Table summarises research gaps to guide the public health response identified.", "The Table summarises research gaps to guide the public health response identified. In conclusion, there are a number of urgent research priorities to inform the public health response to the global spread of 2019-nCoV infections. Establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-nCoV but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations.", "Establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-nCoV but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations. In addressing the research gaps identified here, there is a need for strong collaboration of a competent corps of epidemiological scientists and public health workers who have the flexibility to cope with the surge capacity required, as well as support from laboratories that can deliver on the ever rising demand for diagnostic tests for 2019-nCoV and related sequelae. The readiness survey by Reusken et al.", "The readiness survey by Reusken et al. in this issue of Eurosurveillance testifies to the rapid response and capabilities of laboratories across Europe should the outbreak originating in Wuhan reach this continent [23] . In the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally.", "In the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally. Beyond the first year, one interesting possibility in the longer term, perhaps borne of wishful hope, is that after the first few epidemic waves, the subsequent endemic re-infections could be of milder severity. Particularly if children are being infected and are developing immunity hereafter, 2019-nCoV could optimistically become the fifth human coronavirus causing the common cold.", "Particularly if children are being infected and are developing immunity hereafter, 2019-nCoV could optimistically become the fifth human coronavirus causing the common cold. None declared." ]

[ "Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2]. The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number.", "Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid- to late-January. Average delays between infection and illness onset have been estimated at around 5–6 days, with an upper limit of around 11-14 days [2,5], and delays from illness onset to laboratory confirmation added a further 10 days on average [2].", "Average delays between infection and illness onset have been estimated at around 5–6 days, with an upper limit of around 11-14 days [2,5], and delays from illness onset to laboratory confirmation added a further 10 days on average [2]. Text: It is now 6 weeks since Chinese health authorities announced the discovery of a novel coronavirus (2019-nCoV) [1] causing a cluster of pneumonia cases in Wuhan, the major transport hub of central China. The earliest human infections had occurred by early December 2019, and a large wet market in central Wuhan was linked to most, but not all, of the initial cases [2] .", "The earliest human infections had occurred by early December 2019, and a large wet market in central Wuhan was linked to most, but not all, of the initial cases [2] . While evidence from the initial outbreak investigations seemed to suggest that 2019-nCoV could not easily spread between humans [3] , it is now very clear that infections have been spreading from person to person [2] . We recently estimated that more than 75,000 infections may have occurred in Wuhan as at 25 January 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland China and around the world.", "We recently estimated that more than 75,000 infections may have occurred in Wuhan as at 25 January 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland China and around the world. A number of important characteristics of 2019-nCoV infection have already been identified, but in order to calibrate public health responses we need improved information on transmission dynamics, severity of the disease, immunity, and the impact of control and mitigation measures that have been applied to date. Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] .", "Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] . The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number.", "Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid-to late-January. Average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] .", "Average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] . Chains of transmission have now been reported in a number of locations outside of mainland China. Within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of Hubei province in China, or in other countries.", "Within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of Hubei province in China, or in other countries. If sustained transmission does occur in other locations, it would be valuable to determine whether there is variation in transmissibility by location, for example because of different behaviours or control measures, or because of different environmental conditions. To address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-nCoV transmission to occur.", "To address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-nCoV transmission to occur. In an analysis of the first 425 confirmed cases of infection, 73% of cases with illness onset between 12 and 22 January reported no exposure to either a wet market or another person with symptoms of a respiratory illness [2] . The lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but China's health minister publicly warned that pre-symptomatic transmission could be occurring [6] .", "The lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but China's health minister publicly warned that pre-symptomatic transmission could be occurring [6] . Determining the extent to which asymptomatic or pre-symptomatic transmission might be occurring is an urgent priority, because it has direct implications for public health and hospital infection control. Data on viral shedding dynamics could help in assessing duration of infectiousness.", "Data on viral shedding dynamics could help in assessing duration of infectiousness. For severe acute respiratory syndrome-related coronavirus (SARS-CoV), infectivity peaked at around 10 days after illness onset [7] , consistent with the peak in viral load at around that time [8] . This allowed control of the SARS epidemic through prompt detection of cases and strict isolation.", "This allowed control of the SARS epidemic through prompt detection of cases and strict isolation. For influenza virus infections, virus shedding is highest on the day of illness onset and relatively higher from shortly before symptom onset until a few days after onset [9] . To date, transmission patterns of 2019-nCoV appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than SARS.", "To date, transmission patterns of 2019-nCoV appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than SARS. Transmission of respiratory viruses generally happens through large respiratory droplets, but some respiratory viruses can spread through fine particle aerosols [10] , and indirect transmission via fomites can also play a role. Coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance.", "Coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance. The SARS-CoV superspreading event at Amoy Gardens where more than 300 cases were infected was attributed to faecal-oral, then airborne, spread through pressure differentials between contaminated effluent pipes, bathroom floor drains and flushing toilets [13] . The first large identifiable superspreading event during the present 2019-nCoV outbreak has apparently taken place on the Diamond Princess cruise liner quarantined off the coast of Yokohama, Japan, with at least 130 passengers tested positive for 2019-nCoV as at 10 February 2020 [14] .", "The first large identifiable superspreading event during the present 2019-nCoV outbreak has apparently taken place on the Diamond Princess cruise liner quarantined off the coast of Yokohama, Japan, with at least 130 passengers tested positive for 2019-nCoV as at 10 February 2020 [14] . Identifying which modes are important for 2019-nCoV transmission would inform the importance of personal protective measures such as face masks (and specifically which types) and hand hygiene. The first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had Modelling studies incorporating healthcare capacity and processes pneumonia.", "The first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had Modelling studies incorporating healthcare capacity and processes pneumonia. It is well established that some infections can be severe, particularly in older adults with underlying medical conditions [15, 16] , but based on the generally mild clinical presentation of 2019-nCoV cases detected outside China, it appears that there could be many more mild infections than severe infections. Determining the spectrum of clinical manifestations of 2019-nCoV infections is perhaps the most urgent research priority, because it determines the strength of public health response required.", "Determining the spectrum of clinical manifestations of 2019-nCoV infections is perhaps the most urgent research priority, because it determines the strength of public health response required. If the seriousness of infection is similar to the 1918/19 Spanish influenza, and therefore at the upper end of severity scales in influenza pandemic plans, the same responses would be warranted for 2019-nCoV as for the most severe influenza pandemics. If, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly.", "If, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly. Beyond a robust assessment of overall severity, it is also important to determine high risk groups. Infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] .", "Infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] . Those under 18 years are a critical group to study in order to tease out the relative roles of susceptibility vs severity as possible underlying causes for the very rare recorded instances of infection in this age group. Are children protected from infection or do they not fall ill after infection?", "Are children protected from infection or do they not fall ill after infection? If they are naturally immune, which is unlikely, we should understand why; otherwise, even if they do not show symptoms, it is important to know if they shed the virus. Obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity.", "Obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity. Answers to these questions are especially pertinent as basis for decisions on school closure as a social distancing intervention, which can be hugely disruptive not only for students but also because of its knock-on effect for child care and parental duties. Very few children have been confirmed 2019-nCoV cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers.", "Very few children have been confirmed 2019-nCoV cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers. Serosurveys in affected locations could inform this, in addition to truly assessing the clinical severity spectrum. Another question on susceptibility is regarding whether 2019-nCoV infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera.", "Another question on susceptibility is regarding whether 2019-nCoV infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera. Some experts already questioned whether the 2019-nCoV may behave similarly to MERS-CoV in cases exhibiting mild symptoms without eliciting neutralising antibodies [17] . A separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] .", "A separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] . If either of these were to be relevant, the transmission dynamics could become more complex. A wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-nCoV.", "A wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-nCoV. Internationally, the past week has seen an increasing number of countries issue travel advisories or outright entry bans on persons from Hubei province or China as a whole, as well as substantial cuts in flights to and from affected areas out of commercial considerations. Evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions.", "Evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions. If and when local transmission begins in a particular location, a variety of community mitigation measures can be implemented by health authorities to reduce transmission and thus reduce the growth rate of an epidemic, reduce the height of the epidemic peak and the peak demand on healthcare services, as well as reduce the total number of infected persons [21] . A number of social distancing measures have already been implemented in Chinese cities in the past few weeks including school and workplace closures.", "A number of social distancing measures have already been implemented in Chinese cities in the past few weeks including school and workplace closures. It should now be an urgent priority to quantify the effects of these measures and specifically whether they can reduce the effective reproductive number below 1, because this will guide the response strategies in other locations. During the 1918/19 influenza pandemic, cities in the United States, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] .", "During the 1918/19 influenza pandemic, cities in the United States, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] . Similarly to international travel interventions, local social distancing measures should be assessed for their impact and when they could be safely discontinued, albeit in a coordinated and deliberate manner across China such that recrudescence in the epidemic curve is minimised. Mobile telephony global positioning system (GPS) data and location services data from social media providers such as Baidu and Tencent in China could become the first occasion when these data inform outbreak control in real time.", "Mobile telephony global positioning system (GPS) data and location services data from social media providers such as Baidu and Tencent in China could become the first occasion when these data inform outbreak control in real time. At the individual level, surgical face masks have often been a particularly visible image from affected cities in China. Face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons.", "Face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons. However, there is now a shortage of supply of masks in China and elsewhere, and debates are ongoing about their protective value for uninfected persons in the general community. The Table summarises research gaps to guide the public health response identified.", "The Table summarises research gaps to guide the public health response identified. In conclusion, there are a number of urgent research priorities to inform the public health response to the global spread of 2019-nCoV infections. Establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-nCoV but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations.", "Establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-nCoV but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations. In addressing the research gaps identified here, there is a need for strong collaboration of a competent corps of epidemiological scientists and public health workers who have the flexibility to cope with the surge capacity required, as well as support from laboratories that can deliver on the ever rising demand for diagnostic tests for 2019-nCoV and related sequelae. The readiness survey by Reusken et al.", "The readiness survey by Reusken et al. in this issue of Eurosurveillance testifies to the rapid response and capabilities of laboratories across Europe should the outbreak originating in Wuhan reach this continent [23] . In the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally.", "In the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally. Beyond the first year, one interesting possibility in the longer term, perhaps borne of wishful hope, is that after the first few epidemic waves, the subsequent endemic re-infections could be of milder severity. Particularly if children are being infected and are developing immunity hereafter, 2019-nCoV could optimistically become the fifth human coronavirus causing the common cold.", "Particularly if children are being infected and are developing immunity hereafter, 2019-nCoV could optimistically become the fifth human coronavirus causing the common cold. None declared." ]

[ "Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2]. The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number.", "Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid- to late-January. Average delays between infection and illness onset have been estimated at around 5–6 days, with an upper limit of around 11-14 days [2,5], and delays from illness onset to laboratory confirmation added a further 10 days on average [2].", "Average delays between infection and illness onset have been estimated at around 5–6 days, with an upper limit of around 11-14 days [2,5], and delays from illness onset to laboratory confirmation added a further 10 days on average [2]. Text: It is now 6 weeks since Chinese health authorities announced the discovery of a novel coronavirus (2019-nCoV) [1] causing a cluster of pneumonia cases in Wuhan, the major transport hub of central China. The earliest human infections had occurred by early December 2019, and a large wet market in central Wuhan was linked to most, but not all, of the initial cases [2] .", "The earliest human infections had occurred by early December 2019, and a large wet market in central Wuhan was linked to most, but not all, of the initial cases [2] . While evidence from the initial outbreak investigations seemed to suggest that 2019-nCoV could not easily spread between humans [3] , it is now very clear that infections have been spreading from person to person [2] . We recently estimated that more than 75,000 infections may have occurred in Wuhan as at 25 January 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland China and around the world.", "We recently estimated that more than 75,000 infections may have occurred in Wuhan as at 25 January 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland China and around the world. A number of important characteristics of 2019-nCoV infection have already been identified, but in order to calibrate public health responses we need improved information on transmission dynamics, severity of the disease, immunity, and the impact of control and mitigation measures that have been applied to date. Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] .", "Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] . The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number.", "Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid-to late-January. Average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] .", "Average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] . Chains of transmission have now been reported in a number of locations outside of mainland China. Within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of Hubei province in China, or in other countries.", "Within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of Hubei province in China, or in other countries. If sustained transmission does occur in other locations, it would be valuable to determine whether there is variation in transmissibility by location, for example because of different behaviours or control measures, or because of different environmental conditions. To address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-nCoV transmission to occur.", "To address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-nCoV transmission to occur. In an analysis of the first 425 confirmed cases of infection, 73% of cases with illness onset between 12 and 22 January reported no exposure to either a wet market or another person with symptoms of a respiratory illness [2] . The lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but China's health minister publicly warned that pre-symptomatic transmission could be occurring [6] .", "The lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but China's health minister publicly warned that pre-symptomatic transmission could be occurring [6] . Determining the extent to which asymptomatic or pre-symptomatic transmission might be occurring is an urgent priority, because it has direct implications for public health and hospital infection control. Data on viral shedding dynamics could help in assessing duration of infectiousness.", "Data on viral shedding dynamics could help in assessing duration of infectiousness. For severe acute respiratory syndrome-related coronavirus (SARS-CoV), infectivity peaked at around 10 days after illness onset [7] , consistent with the peak in viral load at around that time [8] . This allowed control of the SARS epidemic through prompt detection of cases and strict isolation.", "This allowed control of the SARS epidemic through prompt detection of cases and strict isolation. For influenza virus infections, virus shedding is highest on the day of illness onset and relatively higher from shortly before symptom onset until a few days after onset [9] . To date, transmission patterns of 2019-nCoV appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than SARS.", "To date, transmission patterns of 2019-nCoV appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than SARS. Transmission of respiratory viruses generally happens through large respiratory droplets, but some respiratory viruses can spread through fine particle aerosols [10] , and indirect transmission via fomites can also play a role. Coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance.", "Coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance. The SARS-CoV superspreading event at Amoy Gardens where more than 300 cases were infected was attributed to faecal-oral, then airborne, spread through pressure differentials between contaminated effluent pipes, bathroom floor drains and flushing toilets [13] . The first large identifiable superspreading event during the present 2019-nCoV outbreak has apparently taken place on the Diamond Princess cruise liner quarantined off the coast of Yokohama, Japan, with at least 130 passengers tested positive for 2019-nCoV as at 10 February 2020 [14] .", "The first large identifiable superspreading event during the present 2019-nCoV outbreak has apparently taken place on the Diamond Princess cruise liner quarantined off the coast of Yokohama, Japan, with at least 130 passengers tested positive for 2019-nCoV as at 10 February 2020 [14] . Identifying which modes are important for 2019-nCoV transmission would inform the importance of personal protective measures such as face masks (and specifically which types) and hand hygiene. The first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had Modelling studies incorporating healthcare capacity and processes pneumonia.", "The first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had Modelling studies incorporating healthcare capacity and processes pneumonia. It is well established that some infections can be severe, particularly in older adults with underlying medical conditions [15, 16] , but based on the generally mild clinical presentation of 2019-nCoV cases detected outside China, it appears that there could be many more mild infections than severe infections. Determining the spectrum of clinical manifestations of 2019-nCoV infections is perhaps the most urgent research priority, because it determines the strength of public health response required.", "Determining the spectrum of clinical manifestations of 2019-nCoV infections is perhaps the most urgent research priority, because it determines the strength of public health response required. If the seriousness of infection is similar to the 1918/19 Spanish influenza, and therefore at the upper end of severity scales in influenza pandemic plans, the same responses would be warranted for 2019-nCoV as for the most severe influenza pandemics. If, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly.", "If, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly. Beyond a robust assessment of overall severity, it is also important to determine high risk groups. Infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] .", "Infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] . Those under 18 years are a critical group to study in order to tease out the relative roles of susceptibility vs severity as possible underlying causes for the very rare recorded instances of infection in this age group. Are children protected from infection or do they not fall ill after infection?", "Are children protected from infection or do they not fall ill after infection? If they are naturally immune, which is unlikely, we should understand why; otherwise, even if they do not show symptoms, it is important to know if they shed the virus. Obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity.", "Obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity. Answers to these questions are especially pertinent as basis for decisions on school closure as a social distancing intervention, which can be hugely disruptive not only for students but also because of its knock-on effect for child care and parental duties. Very few children have been confirmed 2019-nCoV cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers.", "Very few children have been confirmed 2019-nCoV cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers. Serosurveys in affected locations could inform this, in addition to truly assessing the clinical severity spectrum. Another question on susceptibility is regarding whether 2019-nCoV infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera.", "Another question on susceptibility is regarding whether 2019-nCoV infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera. Some experts already questioned whether the 2019-nCoV may behave similarly to MERS-CoV in cases exhibiting mild symptoms without eliciting neutralising antibodies [17] . A separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] .", "A separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] . If either of these were to be relevant, the transmission dynamics could become more complex. A wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-nCoV.", "A wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-nCoV. Internationally, the past week has seen an increasing number of countries issue travel advisories or outright entry bans on persons from Hubei province or China as a whole, as well as substantial cuts in flights to and from affected areas out of commercial considerations. Evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions.", "Evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions. If and when local transmission begins in a particular location, a variety of community mitigation measures can be implemented by health authorities to reduce transmission and thus reduce the growth rate of an epidemic, reduce the height of the epidemic peak and the peak demand on healthcare services, as well as reduce the total number of infected persons [21] . A number of social distancing measures have already been implemented in Chinese cities in the past few weeks including school and workplace closures.", "A number of social distancing measures have already been implemented in Chinese cities in the past few weeks including school and workplace closures. It should now be an urgent priority to quantify the effects of these measures and specifically whether they can reduce the effective reproductive number below 1, because this will guide the response strategies in other locations. During the 1918/19 influenza pandemic, cities in the United States, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] .", "During the 1918/19 influenza pandemic, cities in the United States, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] . Similarly to international travel interventions, local social distancing measures should be assessed for their impact and when they could be safely discontinued, albeit in a coordinated and deliberate manner across China such that recrudescence in the epidemic curve is minimised. Mobile telephony global positioning system (GPS) data and location services data from social media providers such as Baidu and Tencent in China could become the first occasion when these data inform outbreak control in real time.", "Mobile telephony global positioning system (GPS) data and location services data from social media providers such as Baidu and Tencent in China could become the first occasion when these data inform outbreak control in real time. At the individual level, surgical face masks have often been a particularly visible image from affected cities in China. Face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons.", "Face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons. However, there is now a shortage of supply of masks in China and elsewhere, and debates are ongoing about their protective value for uninfected persons in the general community. The Table summarises research gaps to guide the public health response identified.", "The Table summarises research gaps to guide the public health response identified. In conclusion, there are a number of urgent research priorities to inform the public health response to the global spread of 2019-nCoV infections. Establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-nCoV but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations.", "Establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-nCoV but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations. In addressing the research gaps identified here, there is a need for strong collaboration of a competent corps of epidemiological scientists and public health workers who have the flexibility to cope with the surge capacity required, as well as support from laboratories that can deliver on the ever rising demand for diagnostic tests for 2019-nCoV and related sequelae. The readiness survey by Reusken et al.", "The readiness survey by Reusken et al. in this issue of Eurosurveillance testifies to the rapid response and capabilities of laboratories across Europe should the outbreak originating in Wuhan reach this continent [23] . In the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally.", "In the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally. Beyond the first year, one interesting possibility in the longer term, perhaps borne of wishful hope, is that after the first few epidemic waves, the subsequent endemic re-infections could be of milder severity. Particularly if children are being infected and are developing immunity hereafter, 2019-nCoV could optimistically become the fifth human coronavirus causing the common cold.", "Particularly if children are being infected and are developing immunity hereafter, 2019-nCoV could optimistically become the fifth human coronavirus causing the common cold. None declared." ]

[ "Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2]. The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number.", "Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid- to late-January. Average delays between infection and illness onset have been estimated at around 5–6 days, with an upper limit of around 11-14 days [2,5], and delays from illness onset to laboratory confirmation added a further 10 days on average [2].", "Average delays between infection and illness onset have been estimated at around 5–6 days, with an upper limit of around 11-14 days [2,5], and delays from illness onset to laboratory confirmation added a further 10 days on average [2]. Text: It is now 6 weeks since Chinese health authorities announced the discovery of a novel coronavirus (2019-nCoV) [1] causing a cluster of pneumonia cases in Wuhan, the major transport hub of central China. The earliest human infections had occurred by early December 2019, and a large wet market in central Wuhan was linked to most, but not all, of the initial cases [2] .", "The earliest human infections had occurred by early December 2019, and a large wet market in central Wuhan was linked to most, but not all, of the initial cases [2] . While evidence from the initial outbreak investigations seemed to suggest that 2019-nCoV could not easily spread between humans [3] , it is now very clear that infections have been spreading from person to person [2] . We recently estimated that more than 75,000 infections may have occurred in Wuhan as at 25 January 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland China and around the world.", "We recently estimated that more than 75,000 infections may have occurred in Wuhan as at 25 January 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland China and around the world. A number of important characteristics of 2019-nCoV infection have already been identified, but in order to calibrate public health responses we need improved information on transmission dynamics, severity of the disease, immunity, and the impact of control and mitigation measures that have been applied to date. Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] .", "Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] . The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number.", "Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid-to late-January. Average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] .", "Average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] . Chains of transmission have now been reported in a number of locations outside of mainland China. Within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of Hubei province in China, or in other countries.", "Within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of Hubei province in China, or in other countries. If sustained transmission does occur in other locations, it would be valuable to determine whether there is variation in transmissibility by location, for example because of different behaviours or control measures, or because of different environmental conditions. To address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-nCoV transmission to occur.", "To address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-nCoV transmission to occur. In an analysis of the first 425 confirmed cases of infection, 73% of cases with illness onset between 12 and 22 January reported no exposure to either a wet market or another person with symptoms of a respiratory illness [2] . The lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but China's health minister publicly warned that pre-symptomatic transmission could be occurring [6] .", "The lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but China's health minister publicly warned that pre-symptomatic transmission could be occurring [6] . Determining the extent to which asymptomatic or pre-symptomatic transmission might be occurring is an urgent priority, because it has direct implications for public health and hospital infection control. Data on viral shedding dynamics could help in assessing duration of infectiousness.", "Data on viral shedding dynamics could help in assessing duration of infectiousness. For severe acute respiratory syndrome-related coronavirus (SARS-CoV), infectivity peaked at around 10 days after illness onset [7] , consistent with the peak in viral load at around that time [8] . This allowed control of the SARS epidemic through prompt detection of cases and strict isolation.", "This allowed control of the SARS epidemic through prompt detection of cases and strict isolation. For influenza virus infections, virus shedding is highest on the day of illness onset and relatively higher from shortly before symptom onset until a few days after onset [9] . To date, transmission patterns of 2019-nCoV appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than SARS.", "To date, transmission patterns of 2019-nCoV appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than SARS. Transmission of respiratory viruses generally happens through large respiratory droplets, but some respiratory viruses can spread through fine particle aerosols [10] , and indirect transmission via fomites can also play a role. Coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance.", "Coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance. The SARS-CoV superspreading event at Amoy Gardens where more than 300 cases were infected was attributed to faecal-oral, then airborne, spread through pressure differentials between contaminated effluent pipes, bathroom floor drains and flushing toilets [13] . The first large identifiable superspreading event during the present 2019-nCoV outbreak has apparently taken place on the Diamond Princess cruise liner quarantined off the coast of Yokohama, Japan, with at least 130 passengers tested positive for 2019-nCoV as at 10 February 2020 [14] .", "The first large identifiable superspreading event during the present 2019-nCoV outbreak has apparently taken place on the Diamond Princess cruise liner quarantined off the coast of Yokohama, Japan, with at least 130 passengers tested positive for 2019-nCoV as at 10 February 2020 [14] . Identifying which modes are important for 2019-nCoV transmission would inform the importance of personal protective measures such as face masks (and specifically which types) and hand hygiene. The first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had Modelling studies incorporating healthcare capacity and processes pneumonia.", "The first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had Modelling studies incorporating healthcare capacity and processes pneumonia. It is well established that some infections can be severe, particularly in older adults with underlying medical conditions [15, 16] , but based on the generally mild clinical presentation of 2019-nCoV cases detected outside China, it appears that there could be many more mild infections than severe infections. Determining the spectrum of clinical manifestations of 2019-nCoV infections is perhaps the most urgent research priority, because it determines the strength of public health response required.", "Determining the spectrum of clinical manifestations of 2019-nCoV infections is perhaps the most urgent research priority, because it determines the strength of public health response required. If the seriousness of infection is similar to the 1918/19 Spanish influenza, and therefore at the upper end of severity scales in influenza pandemic plans, the same responses would be warranted for 2019-nCoV as for the most severe influenza pandemics. If, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly.", "If, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly. Beyond a robust assessment of overall severity, it is also important to determine high risk groups. Infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] .", "Infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] . Those under 18 years are a critical group to study in order to tease out the relative roles of susceptibility vs severity as possible underlying causes for the very rare recorded instances of infection in this age group. Are children protected from infection or do they not fall ill after infection?", "Are children protected from infection or do they not fall ill after infection? If they are naturally immune, which is unlikely, we should understand why; otherwise, even if they do not show symptoms, it is important to know if they shed the virus. Obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity.", "Obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity. Answers to these questions are especially pertinent as basis for decisions on school closure as a social distancing intervention, which can be hugely disruptive not only for students but also because of its knock-on effect for child care and parental duties. Very few children have been confirmed 2019-nCoV cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers.", "Very few children have been confirmed 2019-nCoV cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers. Serosurveys in affected locations could inform this, in addition to truly assessing the clinical severity spectrum. Another question on susceptibility is regarding whether 2019-nCoV infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera.", "Another question on susceptibility is regarding whether 2019-nCoV infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera. Some experts already questioned whether the 2019-nCoV may behave similarly to MERS-CoV in cases exhibiting mild symptoms without eliciting neutralising antibodies [17] . A separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] .", "A separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] . If either of these were to be relevant, the transmission dynamics could become more complex. A wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-nCoV.", "A wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-nCoV. Internationally, the past week has seen an increasing number of countries issue travel advisories or outright entry bans on persons from Hubei province or China as a whole, as well as substantial cuts in flights to and from affected areas out of commercial considerations. Evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions.", "Evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions. If and when local transmission begins in a particular location, a variety of community mitigation measures can be implemented by health authorities to reduce transmission and thus reduce the growth rate of an epidemic, reduce the height of the epidemic peak and the peak demand on healthcare services, as well as reduce the total number of infected persons [21] . A number of social distancing measures have already been implemented in Chinese cities in the past few weeks including school and workplace closures.", "A number of social distancing measures have already been implemented in Chinese cities in the past few weeks including school and workplace closures. It should now be an urgent priority to quantify the effects of these measures and specifically whether they can reduce the effective reproductive number below 1, because this will guide the response strategies in other locations. During the 1918/19 influenza pandemic, cities in the United States, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] .", "During the 1918/19 influenza pandemic, cities in the United States, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] . Similarly to international travel interventions, local social distancing measures should be assessed for their impact and when they could be safely discontinued, albeit in a coordinated and deliberate manner across China such that recrudescence in the epidemic curve is minimised. Mobile telephony global positioning system (GPS) data and location services data from social media providers such as Baidu and Tencent in China could become the first occasion when these data inform outbreak control in real time.", "Mobile telephony global positioning system (GPS) data and location services data from social media providers such as Baidu and Tencent in China could become the first occasion when these data inform outbreak control in real time. At the individual level, surgical face masks have often been a particularly visible image from affected cities in China. Face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons.", "Face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons. However, there is now a shortage of supply of masks in China and elsewhere, and debates are ongoing about their protective value for uninfected persons in the general community. The Table summarises research gaps to guide the public health response identified.", "The Table summarises research gaps to guide the public health response identified. In conclusion, there are a number of urgent research priorities to inform the public health response to the global spread of 2019-nCoV infections. Establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-nCoV but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations.", "Establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-nCoV but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations. In addressing the research gaps identified here, there is a need for strong collaboration of a competent corps of epidemiological scientists and public health workers who have the flexibility to cope with the surge capacity required, as well as support from laboratories that can deliver on the ever rising demand for diagnostic tests for 2019-nCoV and related sequelae. The readiness survey by Reusken et al.", "The readiness survey by Reusken et al. in this issue of Eurosurveillance testifies to the rapid response and capabilities of laboratories across Europe should the outbreak originating in Wuhan reach this continent [23] . In the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally.", "In the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally. Beyond the first year, one interesting possibility in the longer term, perhaps borne of wishful hope, is that after the first few epidemic waves, the subsequent endemic re-infections could be of milder severity. Particularly if children are being infected and are developing immunity hereafter, 2019-nCoV could optimistically become the fifth human coronavirus causing the common cold.", "Particularly if children are being infected and are developing immunity hereafter, 2019-nCoV could optimistically become the fifth human coronavirus causing the common cold. None declared." ]

[ "Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2]. The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number.", "Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid- to late-January. Average delays between infection and illness onset have been estimated at around 5–6 days, with an upper limit of around 11-14 days [2,5], and delays from illness onset to laboratory confirmation added a further 10 days on average [2].", "Average delays between infection and illness onset have been estimated at around 5–6 days, with an upper limit of around 11-14 days [2,5], and delays from illness onset to laboratory confirmation added a further 10 days on average [2]. Text: It is now 6 weeks since Chinese health authorities announced the discovery of a novel coronavirus (2019-nCoV) [1] causing a cluster of pneumonia cases in Wuhan, the major transport hub of central China. The earliest human infections had occurred by early December 2019, and a large wet market in central Wuhan was linked to most, but not all, of the initial cases [2] .", "The earliest human infections had occurred by early December 2019, and a large wet market in central Wuhan was linked to most, but not all, of the initial cases [2] . While evidence from the initial outbreak investigations seemed to suggest that 2019-nCoV could not easily spread between humans [3] , it is now very clear that infections have been spreading from person to person [2] . We recently estimated that more than 75,000 infections may have occurred in Wuhan as at 25 January 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland China and around the world.", "We recently estimated that more than 75,000 infections may have occurred in Wuhan as at 25 January 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland China and around the world. A number of important characteristics of 2019-nCoV infection have already been identified, but in order to calibrate public health responses we need improved information on transmission dynamics, severity of the disease, immunity, and the impact of control and mitigation measures that have been applied to date. Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] .", "Infections with 2019-nCoV can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] . The serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. Control measures and changes in population behaviour later in January should have reduced the effective reproductive number.", "Control measures and changes in population behaviour later in January should have reduced the effective reproductive number. However, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid-to late-January. Average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] .", "Average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] . Chains of transmission have now been reported in a number of locations outside of mainland China. Within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of Hubei province in China, or in other countries.", "Within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of Hubei province in China, or in other countries. If sustained transmission does occur in other locations, it would be valuable to determine whether there is variation in transmissibility by location, for example because of different behaviours or control measures, or because of different environmental conditions. To address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-nCoV transmission to occur.", "To address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-nCoV transmission to occur. In an analysis of the first 425 confirmed cases of infection, 73% of cases with illness onset between 12 and 22 January reported no exposure to either a wet market or another person with symptoms of a respiratory illness [2] . The lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but China's health minister publicly warned that pre-symptomatic transmission could be occurring [6] .", "The lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but China's health minister publicly warned that pre-symptomatic transmission could be occurring [6] . Determining the extent to which asymptomatic or pre-symptomatic transmission might be occurring is an urgent priority, because it has direct implications for public health and hospital infection control. Data on viral shedding dynamics could help in assessing duration of infectiousness.", "Data on viral shedding dynamics could help in assessing duration of infectiousness. For severe acute respiratory syndrome-related coronavirus (SARS-CoV), infectivity peaked at around 10 days after illness onset [7] , consistent with the peak in viral load at around that time [8] . This allowed control of the SARS epidemic through prompt detection of cases and strict isolation.", "This allowed control of the SARS epidemic through prompt detection of cases and strict isolation. For influenza virus infections, virus shedding is highest on the day of illness onset and relatively higher from shortly before symptom onset until a few days after onset [9] . To date, transmission patterns of 2019-nCoV appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than SARS.", "To date, transmission patterns of 2019-nCoV appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than SARS. Transmission of respiratory viruses generally happens through large respiratory droplets, but some respiratory viruses can spread through fine particle aerosols [10] , and indirect transmission via fomites can also play a role. Coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance.", "Coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance. The SARS-CoV superspreading event at Amoy Gardens where more than 300 cases were infected was attributed to faecal-oral, then airborne, spread through pressure differentials between contaminated effluent pipes, bathroom floor drains and flushing toilets [13] . The first large identifiable superspreading event during the present 2019-nCoV outbreak has apparently taken place on the Diamond Princess cruise liner quarantined off the coast of Yokohama, Japan, with at least 130 passengers tested positive for 2019-nCoV as at 10 February 2020 [14] .", "The first large identifiable superspreading event during the present 2019-nCoV outbreak has apparently taken place on the Diamond Princess cruise liner quarantined off the coast of Yokohama, Japan, with at least 130 passengers tested positive for 2019-nCoV as at 10 February 2020 [14] . Identifying which modes are important for 2019-nCoV transmission would inform the importance of personal protective measures such as face masks (and specifically which types) and hand hygiene. The first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had Modelling studies incorporating healthcare capacity and processes pneumonia.", "The first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had Modelling studies incorporating healthcare capacity and processes pneumonia. It is well established that some infections can be severe, particularly in older adults with underlying medical conditions [15, 16] , but based on the generally mild clinical presentation of 2019-nCoV cases detected outside China, it appears that there could be many more mild infections than severe infections. Determining the spectrum of clinical manifestations of 2019-nCoV infections is perhaps the most urgent research priority, because it determines the strength of public health response required.", "Determining the spectrum of clinical manifestations of 2019-nCoV infections is perhaps the most urgent research priority, because it determines the strength of public health response required. If the seriousness of infection is similar to the 1918/19 Spanish influenza, and therefore at the upper end of severity scales in influenza pandemic plans, the same responses would be warranted for 2019-nCoV as for the most severe influenza pandemics. If, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly.", "If, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly. Beyond a robust assessment of overall severity, it is also important to determine high risk groups. Infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] .", "Infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] . Those under 18 years are a critical group to study in order to tease out the relative roles of susceptibility vs severity as possible underlying causes for the very rare recorded instances of infection in this age group. Are children protected from infection or do they not fall ill after infection?", "Are children protected from infection or do they not fall ill after infection? If they are naturally immune, which is unlikely, we should understand why; otherwise, even if they do not show symptoms, it is important to know if they shed the virus. Obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity.", "Obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity. Answers to these questions are especially pertinent as basis for decisions on school closure as a social distancing intervention, which can be hugely disruptive not only for students but also because of its knock-on effect for child care and parental duties. Very few children have been confirmed 2019-nCoV cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers.", "Very few children have been confirmed 2019-nCoV cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers. Serosurveys in affected locations could inform this, in addition to truly assessing the clinical severity spectrum. Another question on susceptibility is regarding whether 2019-nCoV infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera.", "Another question on susceptibility is regarding whether 2019-nCoV infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera. Some experts already questioned whether the 2019-nCoV may behave similarly to MERS-CoV in cases exhibiting mild symptoms without eliciting neutralising antibodies [17] . A separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] .", "A separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] . If either of these were to be relevant, the transmission dynamics could become more complex. A wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-nCoV.", "A wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-nCoV. Internationally, the past week has seen an increasing number of countries issue travel advisories or outright entry bans on persons from Hubei province or China as a whole, as well as substantial cuts in flights to and from affected areas out of commercial considerations. Evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions.", "Evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions. If and when local transmission begins in a particular location, a variety of community mitigation measures can be implemented by health authorities to reduce transmission and thus reduce the growth rate of an epidemic, reduce the height of the epidemic peak and the peak demand on healthcare services, as well as reduce the total number of infected persons [21] . A number of social distancing measures have already been implemented in Chinese cities in the past few weeks including school and workplace closures.", "A number of social distancing measures have already been implemented in Chinese cities in the past few weeks including school and workplace closures. It should now be an urgent priority to quantify the effects of these measures and specifically whether they can reduce the effective reproductive number below 1, because this will guide the response strategies in other locations. During the 1918/19 influenza pandemic, cities in the United States, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] .", "During the 1918/19 influenza pandemic, cities in the United States, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] . Similarly to international travel interventions, local social distancing measures should be assessed for their impact and when they could be safely discontinued, albeit in a coordinated and deliberate manner across China such that recrudescence in the epidemic curve is minimised. Mobile telephony global positioning system (GPS) data and location services data from social media providers such as Baidu and Tencent in China could become the first occasion when these data inform outbreak control in real time.", "Mobile telephony global positioning system (GPS) data and location services data from social media providers such as Baidu and Tencent in China could become the first occasion when these data inform outbreak control in real time. At the individual level, surgical face masks have often been a particularly visible image from affected cities in China. Face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons.", "Face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons. However, there is now a shortage of supply of masks in China and elsewhere, and debates are ongoing about their protective value for uninfected persons in the general community. The Table summarises research gaps to guide the public health response identified.", "The Table summarises research gaps to guide the public health response identified. In conclusion, there are a number of urgent research priorities to inform the public health response to the global spread of 2019-nCoV infections. Establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-nCoV but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations.", "Establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-nCoV but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations. In addressing the research gaps identified here, there is a need for strong collaboration of a competent corps of epidemiological scientists and public health workers who have the flexibility to cope with the surge capacity required, as well as support from laboratories that can deliver on the ever rising demand for diagnostic tests for 2019-nCoV and related sequelae. The readiness survey by Reusken et al.", "The readiness survey by Reusken et al. in this issue of Eurosurveillance testifies to the rapid response and capabilities of laboratories across Europe should the outbreak originating in Wuhan reach this continent [23] . In the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally.", "In the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally. Beyond the first year, one interesting possibility in the longer term, perhaps borne of wishful hope, is that after the first few epidemic waves, the subsequent endemic re-infections could be of milder severity. Particularly if children are being infected and are developing immunity hereafter, 2019-nCoV could optimistically become the fifth human coronavirus causing the common cold.", "Particularly if children are being infected and are developing immunity hereafter, 2019-nCoV could optimistically become the fifth human coronavirus causing the common cold. None declared." ]