Datasets:

CShorten

/

1000-CORD19-Papers-Text

like 2

Smallpox was largely a disease of close contacts [17, 26, 33] , spread primarily through face to face contact with an infected person (or occasionally through contaminated clothing). Individuals in the incubation period of smallpox were not infectious, and long term carriers did not exist. Patients were believed to be infectious following the development of oropharyngeal lesions, which could precede the rash by 24 hours [26] . However, patients were believed to be most infectious during the first week of the rash [26] ; Dixon (1962) believed that patients could be infectious from the onset of acute viremia, but most evidence suggested that little transmission occurred prior to the development of the rash [26, 33] . The more severe the case, the more infectious they appeared to be [34] ; mild cases were believed to have very little infectiousness. While scabs contained infectious material and patients were considered to be infectious until the last scab fell off, in practice patients were not highly infectious during the scabbing phase. Importantly, patients who had been vaccinated were found to cause fewer secondary cases [34] . Very severe cases, such as hemorrhagic or flat smallpox, occasionally resulted in considerable transmission, owing to diagnostic difficulties; mild cases, in which the patient remained ambulant during the course of the disease, could cause considerable spread as well [35, 36] . Within a household or family dwelling, the secondary attack rate of unvaccinated susceptibles depended on the time and place, occasionally below 50% [29] , but often approaching 100% [37] . Drier conditions were often believed to favor transmission [17, 27] , so that lower rates of transmission derived from tropical regions may not be applicable to the temperate zone [38] . The number of secondary cases resulting from a given importation into Europe varied widely [39] , with most importations yielding few cases, but with the occasional large outbreak being seen.

Mathematically, we represent the course of smallpox according to Figure 1 . We distinguish eight epidemiologically relevant states: (1) just following exposure, during which time vaccination could afford complete protection against disease, (2) a period of several days during which vaccination will not prevent disease, but may still reduce the severity of disease, (3) still prior to the development of symptoms, but too late for vaccination, (4) the beginning of the pre-eruptive period, during which the patient exhibits fever, malaise, and possibly other symptoms, but is not yet infectious, (5) a short period prior to the appearance of the rash, during which the appearance of oropharyngeal lesions will permit variola transmission, (6) the first week of the rash, during which time the patient is most infectious, (7) and (8), succeeding stages of the rash, during which time the patient is less infectious. For each of these states, we assume that conditional on surviving, the waiting time until the next stage is chosen from a uniform distribution as indicated in Appendix 2 [see Additional file: 2], except that the incubation period (the time from infection until Stage 4) is derived from estimates of the incubation distribution of smallpox based on importation cases in Europe [26] (see Appendix 2 [see additional file 2] for details). We chose to sample from a uniform distribution as a simple way to ensure a minimum waiting time in each state; many alternatives to this choice are possible.

We simulate the transmission of smallpox on a "smallworlds" network (highly clustered, but with short characteristic path lengths) [40] . Specifically, we assume that each person is located in a single household, and that the transmission rates were greatest in the household. We also assume that a fraction of the population are grouped into workplace or social groups, in which transmission may also occur, but with a lower rate per unit time than for household contacts. Finally, we assume that with a still smaller probability, any individual may transmit infection to any other individual in the population (casual contacts).

In general, in a network-structured model, the number of secondary cases caused by an index case in a completely susceptible population is not a useful index of epidemic potential [41, 42] (for a simple example, see [43] ), since (for instance) an individual could infect everyone in his or her household, and not cause a widespread epidemic unless between-household transmission were sufficiently frequent. Rather than constructing the appropriate generalized basic reproduction number for our model (leading to highly cumbersome expressions), we chose an alternative (ad hoc) index of epidemic potential. For any given scenario of interest, we simulated the introduction of 10 index cases at random into a population of size 10000, and operationally defined "containment" to occur whenever the final size of the epidemic was less than 500 cases within 250 days (we showed, in the discussion of Figure Smallpox stages used in the simulation model Figure 1 Smallpox stages used in the simulation model. Flat and ordinary smallpox rashes are indicated with more dots than modified and "mild" smallpox, suggesting potentially greater infectiousness. Hemorrhagic smallpox is indicated by horizontal line shading. Further details are provided in Table 6 . 5A below, that in nearly all cases, the 250-day window differs very little from a 1000-day window). Because we simulate a disease with a finite duration on a finite and nonrenewing population, epidemic extinction always occurs in finite time.

We assume that even in the absence of specific case investigations, the presence of smallpox symptoms will prompt patients to be diagnosed; we assume, however, a higher diagnosis rate for all forms of ordinary smallpox than for the severe flat and hemorrhagic forms, or for the mildest form. We assume that once an individual is diagnosed, their household and workplace contacts are investigated and detected with some probability; we assume that a high fraction (such as 95%) of household contacts are assumed to be traceable (see below). We assume that the fraction of workplace/social contacts that are traceable is less than the fraction of household contacts that are traceable; we assume that no casual contacts are traceable.

High contact-finding rates may be plausible; we examined San Francisco Department of Public Health records of contact investigations for meningococcal disease (like smallpox, a potentially fatal disease for which rapid intervention may prevent mortality and morbidity). Records were available from December 2001 to April 2002; 13 such investigations during this period resulted in identification of 62 household contacts, all of which were contacted; out of 38 workplace/social contacts identified, 32 were contacted (84%).

In our model, we assume that identified asymptomatic contacts are vaccinated, quarantined, and monitored for symptom development, while symptomatic patients are isolated and treated as necessary [9] ; thus, the modeled interventions include more than ring vaccination alone. Finally, we include the possibility that all contacts (of both symptomatic and asymptomatic) traced and the same procedure applied, so that all contacts of contacts would be investigated. We assume that uninfected or asymptomatic individuals who are visited or traced individuals will be diagnosed more rapidly than if they had not been traced; in fact, such individuals would be isolated and would not be able to continue a chain of transmission. We follow previous models [9] in assuming a limited vaccination capability of K r per day for ring vaccination. We assumed one of two strategies for contact tracing: (1) tracing only of direct contacts of diagnosed cases, and (2) tracing of contacts of contacts of diagnosed cases as well as direct contacts.

The contact structure of the network is illustrated in Figure 2 . Observe that individuals b and c are household contacts of individual a, so that if individual a were a smallpox case, an attempt would be made to find and vaccinate individuals b and c as household contacts of a case. If individuals a and b were both cases, then two attempts could be made to find individual c. We have modeled the effect of multiple contact-finding attempts conservatively in the sense that if the first attempt to find an individual as a household contact (of a case or of a contact) is determined to fail, no further attempts will be made. This maintains the failure rate of contact tracing (looked at from the standpoint of finding individuals) even in large households. Similar considerations apply to workplace/social groups. Figure 2 Network structure shown for households (joined by thick lines) of size 3 and workplace/social groups of size 4 (joined by thin lines); a small portion of the network is shown. Individual a has two household contacts (b and c), and three workplace/social contacts (d, e, and f). If individual a were a smallpox case, the household contacts would be at highest risk for acquiring smallpox, followed by workplace/social contacts; all individuals in the population are at a low risk of casual transmission from individual a. Case investigation of individual a would identify the direct contacts b-f with probabilities that depend on whether the contact is household or workplace/social; if such individuals are identified, they will be vaccinated. If contacts of contacts are being traced, the investigation will subsequently identify individuals g-p.

We analyzed the model in three ways. First, we selected a Latin Hypercube sample [44] [45] [46] of parameters chosen uniformly from the parameter ranges given in Appendix 2 [see additional file 2] , and simulated the transmission and control of smallpox to determine which parameters were most important for contact tracing and ring vaccination to be effective. Second, we used the same Latin Hypercube Sample of input parameters, but assumed that all disease control efforts were inactive. We used these parameters to simulate smallpox transmission, but then iteratively selected transmission parameters so that (1) between 1% and 10% of new infections resulted from casual (random) transmission, and (2) each index case resulted in between two and five secondary cases (thought to be plausible for historic smallpox; [8] suggest three secondary cases). For each of the resulting smallpox parameter sets using 100 stochastic simulations per set, we determined the daily ring vaccination/case tracking capacity needed to contain all simulated smallpox epidemics (i.e., keep the total number of cases below 500 within 250 days). Third and finally, we chose parameter values to yield an moderately large smallpox epidemic (with each index case causing approximately six secondary cases), and present illustrative scenarios for ring vaccination. These scenarios are intended to complement the simulations which were calibrated to historic smallpox, since the characteristics of smallpox that may be used in a deliberate release are not known. It is important to realize that in our model, the case finding time determines the fraction of contacts that will become infected, and that our model parameters have been chosen to yield quite rapid transmission to close contacts; in reality, much transmission of natural smallpox occurred through "sickbed" routes which would not occur in a modern setting [47] , so that in this sense our model errs considerably on the side of caution and pessimism.

To determine which of the input parameters were most important in determining the total number of smallpox cases, we selected a Latin Hypercube sample of size 1000 from the input parameter ranges indicated in Appendix 2 [see additional file 2] and simulated the mean number of cases within 250 days in a population of 10000. We then computed the partial rank correlation coefficient [46] (PRCC; see Appendix 2 [see additional file 2]) between each input parameter and the number of smallpox cases; when the PRCC is close to zero, the value of the parameter has little relation to the simulation output; when the PRCC is close to +1.0 or -1.0, the value of the parameter is highly important in determining the simulation output. Neglecting the number of index cases (which is directly related to the number of new cases), those parameters whose PRCC exceeds 0.1 are shown in Table 2 . Most of these parameters identified as important are related to the density of available contacts (mean household size, prior vaccination fraction, and protection due to prior vaccination) or the transmission rate and infectivity (including the length of the pre-eruptive infectious period (stage 5 in Figure 2 )). Note, however, that the speed of ring vaccination (household tracing delay) and faster diagnosis due to awareness of the outbreak are important parameters. Additionally, the infectivity of mild cases appears as an important parameter as well.

To explore factors which contribute to the success of ring vaccination, we chose smallpox scenarios which resulted in severe and fast-moving epidemics in the absence of disease control; these simulated epidemics are considerably more severe than is believed likely under present circumstances.

We used these parameters to simulate smallpox epidemics beginning with 10 cases, for a variety of levels of ring vaccination capacity per day (contact tracing capacity per day), as shown in Figure 3A . In this Figure, we assume that the population size is 10000, and that the epidemic began with 10 infected individuals. The mean household size is assumed to be 4, the mean size of the workplace/social contact group is 8, and contacts of contacts are traced. We assume that each day, the number of contacts that can be traced and vaccinated as a result of case investigation is 0, 10, 20, 30 and 40 per day; the probability of finding a workplace/social contact is assumed to be 80%. The Table 1 .

Because we assumed nonzero diagnosis probabilities during the prodromal period for all individuals in Figure 3A , we repeated the simulation assuming no diagnosis in the prodromal period unless individuals were under specific surveillance. The results were nearly identical: assuming 30 contact tracings (ring vaccinations) per day, we found 26% of the scenarios in Figure 3A exhibited decontainment, and 28% assuming no diagnosis during the prodromal period; assuming 40 contact tracings per day, we found 1 out of 100 scenarios showed loss of containment in Figure 3A and when we repeated the scenario of Figure 3A assuming no diagnosis during the prodromal period.

In Figure 3B , we illustrate control of an epidemic for which all parameters are identical to Figure 3A , except that the workplace/social group size is 12 (instead of 8, as in Figure 3A ), and the probability of finding workplace/ social contacts is 60% (instead of 80%, as in Figure 3A ). In this case, the larger size of the workplace/social groups and the lower contact finding probability makes it necessary to have a higher ring vaccination capacity to attain a high probability of containing the epidemic, and on average it takes longer for eradication to finally occur.

Finally, in Figure 3C , we show control of an epidemic in a population of 100,000, beginning with 1000 initial infectives, keeping all other parameters the same as in Figure 3A . Each curve corresponds to the indicated number of possible ring vaccinations per day. This figure shows that assuming sufficient capacity, ring vaccination is in principle capable of containing even epidemics beginning with very many infected individuals. However, mass vaccination in such cases is justified because of the far larger number of individuals at risk and the inability to perform such extensive contact tracing.

In Figure 3D , we compare the effect of tracing contacts of contacts (as described in Appendix 2 [see additional file 2]) at different levels of ring vaccination capacity. Thin Figure 5A , 5B φ

Prob. of finding household contact 0.95 Delay, tracing household contacts 1-5 days Expanding severe smallpox epidemic Figure 3 3A -Expanding severe smallpox epidemic beginning with 10 initial cases, assuming 0, 10, 20, 30, and 40 possible ring vaccinations per day. The household size is 4 and the workplace/social group size is 8; we assume 95% of household contacts are traceable (with a mean delay of 1 day) and 80% of workplace/social contacts are traceable (with a mean delay of 2 days). We also assume that 25% of the population have 50% protection from infection resulting from vaccination prior to the discontinuation of routine vaccination. We assume that infection will be transmitted to close contacts with a mean time of 0.2 days, and that each person while infective causes on average 0.15 casual (untraceable) infections per day. We assume that individuals are 20% as infectious in the day just before the appearance of the rash as they will be during the first week of the rash, and that individuals are 20% as infectious as this (4% as infectious as during the first week of the rash) during the prodromal period. We assume that diagnosis rates will increase by a factor of 50% after smallpox becomes known to the community; we assume that each individual contacted during an investigation has a additional diagnosis or removal rate of 0.75 per day following the onset of symptoms (reflecting enhanced surveillance or contact isolation). Important parameters are summarized in Table 1 ; the full set of parameter choices is outlined in Tables 8-11 in Appendix 2 [see additional file 2] . Diagnosis times are discussed in Appendix 2 [see additional file 2]. 3B -An expanding severe smallpox epidemic under inadequate ring vaccination is shown for parameters identical to Figure 3A , except that workplace/social group sizes are 12 (instead of 8), and the probability of tracing workplace/social contacts is 0.6 (instead of 0.8).

3C -A severe smallpox epidemic is controlled by ring vaccination despite the large number of initial cases. The parameters are identical to Figure 3A , except that 1000 index cases inaugurate the attack in these scenarios (and ring vaccination capacity is much greater, as indicated). While not recommended, ring vaccination may ultimately halt epidemics beginning with many index cases if sufficient vaccination capacity were available, contact finding feasible, and follow-up sufficient. 3D -Tracing contacts of contacts (red) is beneficial when sufficient contact tracing/ring vaccination capacity exists (dotted lines). In these scenarios, all parameters are the same as in Figure 3A ; the number of contact tracings possible per day is either 20 or 40 per day. Contacts of contacts are traced in two scenarios; in the other two, only direct contacts of cases are traced. For low levels of ring vaccination (20 per day), tracing contacts of contacts is harmful; for high levels (40 per day) of ring vaccination, it is beneficial to trace contacts of contacts. When the contact tracing/ring vaccination capacity is too small to adequately cover contacts of the cases themselves, diversion of resources to contacts of contacts is harmful; however, provided that sufficient capacity exists, tracing contacts of contacts helps outrun the chain of transmission. Each line corresponds to the average of 100 realizations. The average number infected on each day is plotted in the Figure. The figure illustrates that when ring vaccination capacity is low, tracing contacts of contacts (as modeled) yields a more severe average epidemic; when ring vaccination capacity is large, tracing contacts of contacts results in a less severe average epidemic; if the contact tracing/ring vaccination capacity is too low to cover adequately the contacts of contacts in addition to the contacts of cases, extension of tracing to the contacts of contacts (the second ring) is harmful; however, if there is sufficient capacity to cover the contacts of contacts, then the tracing of contacts of contacts is beneficial.

Finally, in Figure 4 , we illustrate the considerable variability that may be seen from simulation to simulation. This figure shows twenty simulations when contacts of contacts are not traced. Stochastic variability between realizations is considerable, even when all parameters are held constant; this variability is expected to limit the ability to make inferences based on observation of a single realization of the process.

Because our baseline hazard for infection of individuals may be larger than would be expected for naturally occurring smallpox, we examined the effect of more realistic values of this hazard. In particular, we chose different levels of ring vaccination capacity (10, and 20) , and of the relative hazard for workplace/social contacts, and then chose values of the baseline hazard for infection varying from 0.5 per day (for a mean time to infection of 2 days) to 2 per day (for a mean time to infection of one half day), Table 3 : Estimated decontainment probability for different levels of ring vaccination capacity (Kr) and relative hazard for infection due to workplace/social contacts (h2), for different levels of the baseline hazard for infection from household contacts λ (based on replications of 100 simulations for each level). For each scenario, 10 index cases were introduced into a population of size 10000. All other parameters were the same as for Figure 3A . As before, we define decontainment to mean that the total number of cases from 10 index cases eventually exceeded 500 by day 250. and introduced 10 index cases into a population of 10000. We then repeated this 100 times, and reported the fraction of scenarios in which the number of infections ultimately exceeded 500 (as before, chosen as a cutoff to indicate the ultimate "escape" of containment of the epidemic). These results, shown in Table 3 , support the idea that ring vaccination can easily control introduced smallpox provided there is sufficient capacity and efficacy of tracing.

Because of considerable uncertainty in the model parameters, we chose a collection of parameter values, and for each, estimated the containment probability (operationally defined as fewer than 500 total cases as a result of 10 index cases, within 250 days). We estimated this containment probability by simulating the smallpox epidemic 100 times for the same parameter values, and computing the frequency out of these 100 realizations for which fewer than 500 index cases resulted within 250 days.

(Using a 1000 day window produces slightly smaller containment estimates; for 3 out of 1000 parameter set choices, this difference was greater than 0.06; the maximum difference seen was 0.23; the mean absolute difference was 0.0029; in only one case out of 1000 did we see containment in all 100 cases for the 250-day window, but not in all 100 cases for the 1000-day window).

One thousand scenarios chosen from a Latin Hypercube sample were analyzed, and as indicated before, we chose the hazard for close contact transmission and the hazard for random transmission to guarantee that between 2 and 5 secondary cases per case occur, and that no more than 5% of cases are attributable to random transmission (we refer to this set as the "calibrated" scenarios further in this text). Having chosen this collection of 1000 parameter sets, we considered two levels of two different control parameters which were applied to each (so that each of the 1000 parameter sets were simulated under four different control conditions). The first of the two control parameters was the probability of workplace/social group contact finding; we chose values of 0.8 and 0.9 for this parameter (the household contact finding probability was 0.95 in all cases). The second of the control parameters was the rate of diagnosis (and effective removal) from the community of cases developing among previously identified and traced contacts who were initially asymptomatic (we refer to this as the monitored diagnosis rate); we assumed first a low level corresponding to a mean diagnosis time of one day from the onset of symptoms, and a high level corresponding to a mean time of 3 hours from the onset of symptoms (high levels of the monitored diagnosis rate correspond effectively to isolation of contacts). Finally, we assumed a doubling of the diagnosis rate after the beginning of widespread community awareness of smallpox. We then computed the containment fraction at different levels of ring vaccination capacity (contact tracing capacity per day). Thus, for each of 1000 scenarios (parameter set choices), we assigned the workplace/social group contact tracing success probability (υ 2 ), the monitored diagnosis rate φ (Appendix 2 [see additional file 2]), and the contact tracing/ring vaccination capacity per day (K r ). We then performed 100 realizations beginning with 10 index cases, and computed the containment fraction (fraction showing fewer than 500 cases in 250 days, beginning with 10 index cases). Thus, for each of the two choices each of υ 2 and φ, and for each value of K r we examined, we obtained 1000 values of the containment fraction. We use the resulting distributions in Figure 5A (averaging over these 1000 containment fractions), and Figure 5B (displaying the minimum value of the 1000 containment fractions).

In Figure 5A , we plot the mean containment fraction (averaging the containment fraction over all 1000 scenarios), as ring vaccination capacity varies, for the two levels of workplace/social group contact finding probabilities (0.8 and 0.9), and for the two levels of monitored diagnosis rate among initially asymptomatic contacts (1 day -1 and 8 day -1 ). For low levels of ring vaccination (traceable contacts per day), the epidemic is almost never contained, but for ring vaccination levels near 50-60 per day (5-6 per index case per day), the average containment fraction Figure 4 Stochastic variability is illustrated by plotting the number of infectives over time over multiple replications. In this example, most simulations exhibit rapid containment of smallpox. The mean number of cases (averaging over simulations) is influenced by a small number of simulations exhibiting an uncontained epidemic. The parameters are the same as in Figure 3A , except that contacts of contacts are not traced in these replications.

The mean containment probability Figure 5 5A -The mean containment probability increases as the number of ring vaccinations per day is increased. For this figure, the 1000 "calibrated" parameter sets were chosen, and for each parameter set, 100 realizations were simulated and the fraction of these for which the epidemic was contained to fewer than 500 cases was determined. The average of these 1000 containment fractions is plotted on the vertical axis. We assumed a household contact finding probability of 95% and that the diagnosis rates double after community awareness of the epidemic. We considered high levels of workplace/social (w/s) contact finding (0.9), as well as moderate levels (0.8). We also considered two levels of diagnosis of smallpox among investigated (alerted) contacts: high levels (corresponding to a 3 hour mean delay, indicated by "high contact isolation"), and moderate levels (corresponding to a one day delay, and indicated by "less contact isolation"). The figure shows four such conditions, a. high workplace/social contact finding probability and high contact isolation, b. moderate workplace/social contact finding probability and high contact isolation, c. high workplace/social contact finding probability and less contact isolation, and d. moderate workplace/social contact finding probability and less contact isolation. All other parameter values were chosen from the uncertainty analysis (the 1000 "calibrated" parameter sets). In this figure, "contact isolation" refers to the monitored diagnosis rate, i.e. the rate at which previously asymptomatic contacts who subsequently develop disease will be diagnosed (φ, Table 1 , Table 8 ). 5B -The minimum containment probability out of the same 1000 scenarios chosen in Figure 5A . Whereas in Figure 5A , we averaged the simulated containment frequency (out of 100 realizations for each scenario), in this figure we determined which of the 1000 scenarios led to the lowest containment frequency, and we plotted this single worst (out of 1000) containment frequency, at different levels of ring vaccination capacity, for the same four conditions as in Figure 5A : a. high workplace/ social contact finding probability (0.9) and high contact isolation (effective 3 hour delay following symptoms), b. moderate workplace/social contact finding probability (0.8) and high contact isolation, c. high workplace/social contact finding probability (0.9) and less contact isolation (effective one day delay), and d. moderate workplace/social contact finding probability (0.8) and less contact isolation. All parameters are the same as in Figure 5A (the household contact finding probability is 0.95 for all scenarios, and the diagnosis rates are doubled after the onset of community awareness). In this figure, "contact isolation" refers to the monitored diagnosis rate, i.e. the rate at which previously asymptomatic contacts who subsequently develop disease will be diagnosed (φ, c.

d.

became close to 1. However, this average conceals the fact that for some scenarios (parameter sets chosen from the calibrated uncertainty analysis), control remains difficult or impossible even at high levels of ring vaccination. Therefore, in Figure 5B , we plotted the single lowest containment fraction seen out of the 1000 computed; focusing on the single worst scenarios reveals a different picture, and shows that isolation of asymptomatic contacts and very high probabilities of finding workplace or social contacts would be needed to control smallpox under these most pessimistic parameter choices.

Rapid contact tracing in ring vaccination may play an important role in suppressing the epidemic, since the longer it takes to trace a contact, the less likely the vaccine is to be efficacious, and the more opportunities the infected individual may have to transmit disease before they are finally located, isolated, and vaccinated if appropriate. We illustrate this possibility in Figure 6 by examining the same scenario we showed earlier in Figure 3A (e.g. households of size 4, workplace/social groups of size 8, 95% of household contacts traceable, 80% of workplace/social groups traceable, an average time to infection for a household contact of an infective given by 0.2 days). We assume in one case that contacts may be traced quickly (1 day for a household contact, 2 days for a workplace/ social contact), and in the other that the contacts are on average found slowly (5 days for a household contact, 10 days for workplace/social contacts); we assumed 30 ring vaccinations (traceable contacts) possible per day. In this scenario, the epidemic is more severe and containment (as we have been defining it) less likely when contact tracing is slow: in the fast scenario, 238 infections occurred on average and the (estimated) containment probability was 99%; for the slow scenario, on average 3587 infections occurred and the (estimated) containment probability was only 1%.

While Figure 6 illustrates the possibility that rapid contact tracing may be of decisive importance in some scenarios (parameter set choices), this is not always the case. For some parameter sets, the probability of tracing contacts (household or workplace/social) may be too low, or the transmission rate too high, for more rapid contact tracing to make any difference. Conversely, for other parameter sets, the smallpox transmission rate may be so low that smallpox is easily contained even with slow contact tracing. While rapid contact tracing is never harmful, overall, how typical are the results of Figure 6 (in which rapid contact tracing was important in ensuring the efficacy of ring vaccination)? To address this question, we simulated the growth of smallpox for the 1000 "calibrated" scenarios we used in Figure 5A and 5B. As before, we assumed ten initial cases, and (as in Figure 6 ) that 30 ring vaccinations were possible per day; then we simulated 100 epidemics assuming one day to find a household contact (and 2 days to find a workplace/social contact). We then simulated 100 epidemics assuming that it takes five days to find a household contact and 10 days to find a workplace/social contact (as in Figure 6 ). For each of these 1000 scenarios, we calculated the fraction of simulations for which the total number of cases is 500 or less within 250 days, i.e. the containment fraction. For nearly all scenarios (parameter set choices), the containment fraction was smaller (sometimes much smaller) when the contact finding time is faster (since faster contact finding, all else being equal, improves smallpox control, as illustrated in Figure 6 ). However, for 64.5% of the scenarios (parameter set choices) examined, the difference was less than 2.5% in absolute terms (smallpox was either contained or not contained depending on other factors, and rapid contact tracing did not make the difference). On the other hand, for 18.7% of the scenarios examined, the absolute difference in the containment probability was 20% or more; thus, a substantial difference in containment probability is occa-Faster contact tracing Figure 6 Faster contact tracing may improve the efficacy of ring vaccination. We assume the same baseline parameters as in Figure 3A (e.g. households of size 4, workplace/social groups of size 8, 95% of household contacts traceable, 80% of workplace/social contacts traceable), and 30 ring vaccinations available per day (with contacts of contacts not traced). The fast scenario corresponds to an average one day delay for household and two days for workplace/social contacts (as in Figure 3A ); the slow scenario corresponds to an average five day delay for household and ten day delay for workplace/ social contacts. This figure shows the average of one hundred realizations starting with ten index cases. Effect of more rapid diagnosis Public awareness of smallpox, leading to more rapid isolation and identification, may play an important role in eliminating the epidemic, as illustrated by the scenarios in Figure 7 . We assumed 20 ring vaccinations possible per day, a capacity too small to contain the epidemic in the absence of increased surveillance or diagnosis; the black line in the figure shows the steeply rising average number of cases for the first 100 days. If, however, surveillance or public awareness of the symptoms of smallpox increases the diagnosis rate by 50% (multiplies the baseline diagnosis rates by 1.5), containment becomes possible (blue line); with a doubling of the diagnosis rate (red line) the peak number of cases is lower still. In these scenarios, increased diagnostic rates markedly improve the ability of ring vaccination to control the epidemic, this suggest that any ring vaccination effort be accompanied by increased public awareness and surveillance.

In many cases, however, more rapid diagnosis was not required for ring vaccination to be effective. As before, we simulated smallpox epidemics for each of 1000 calibrated scenarios, performing 100 realizations each beginning with 10 index cases, and computed the fraction of scenarios for which the epidemic was always contained (as defined earlier), assuming no change in diagnosis rates. We assumed 80 ring vaccinators per day, contact finding probabilities of 0.95 for households and 0.8 for workplace/social contacts (as in Figure 3A ). Under these assumptions, for 83.4% of the scenarios, the epidemic was contained within 500 total cases in each of the 100 realizations, even with no change in diagnosis rates. Uncertainty analysis (using the 1000 calibrated scenarios, and based on the fraction of 100 replications showing decontainment) revealed the most important parameters which predict the failure of ring vaccination without more rapid diagnosis were the same as we found in the earlier uncertainty analysis; a higher fraction vaccinated before the epidemic, smaller households or workplace/social groups, less transmissibility, lowered infectivity prior to the rash, more rapid diagnosis, and a higher rate of diagnosis for alerted individuals all contribute to a greater containment probability even without an overall increase in the diagnosis rate.

We have been assuming that whenever an individual is contacted during an investigation, the individual will be diagnosed more quickly should they subsequently develop symptoms. When transmission is assumed to be very rapid (smallpox is assumed to be highly contagious), most individuals may already be infected when identified through contact tracing from an infective. Using the scenario we examined in Figure 3A , we see that continued surveillance of contacts is an essential component of effective ring vaccination designed to control rapidly spreading smallpox: if smallpox in a contact is not diagnosed any more quickly than for a non-contact, containment by ring vaccination requires over 98% contact finding probabilities for both household and workplace/ social contacts -even if unlimited numbers of ring vaccinators are available; containment cannot be guaranteed by adding additional ring vaccination capacity if the contact finding rates are too low and/or the follow-up for contacts is insufficient. Smallpox which is transmitted less rapidly to contacts would, however, be containable with a lower contact finding probability (results not shown).

Finally, we used the "calibrated" scenarios (parameter set choices) to explore the levels of contact finding probability needed to contain the epidemic (as before, defined to mean 500 or fewer cases ultimately resulting from ten initial cases) ( Table 4 ). In these scenarios, we assumed that all traceable contacts were followed up very More rapid diagnosis Figure 7 More rapid diagnosis due to public awareness or increased surveillance may lead to far more effective epidemic control. We assume the same baseline parameters as in Figure 3A , and averaged 100 realizations of the epidemic beginning with 10 index cases and assumed a ring vaccination capacity of 20 per day (and contacts of contacts not traced).

For the black line, the diagnosis rate of cases does not change after the first case is identified (the multiplier is 1.0); for the blue line, the diagnosis rate increases by 50% (multiplier 1.5) after the first case is identified (as in Figure 3A ), resulting in substantially fewer cases; and for the red line, the diagnosis rate is doubled (multiplier 2.0) after the first case is identified, resulting in still fewer cases. quickly (1/a = 1 hour, so that cases arising in previously contacted persons almost never transmit the infection further). We chose different levels of household and workplace/social contact finding probabilities and different levels of ring vaccination capacity, and performed 100 replications of each of the 1000 different scenarios. In Table 4 we report the fraction of scenarios for which all 100 replications exhibited containment. Scenarios in which smallpox is highly contagious require high contact finding probability to ensure the containment of the epidemic.

Transmission prior to the rash makes epidemic control more difficult. In Figure 8 , we show an expanding smallpox epidemic assuming differing levels of infectivity prior to the rash (adding increased infectivity prior to the rash, keeping constant the infectivity after the rash). We assume all parameters are the same as in Figure 3A (and that the ring vaccination capacity is 40 per day). Infectivity prior to the rash is modeled as the relative infectivity during the short (1 day) period of oropharyngeal lesions just prior to the rash (compared to the infectivity during the first week of the rash), and as the relative infectivity during the prodromal period (relative to the period just prior to the rash). We consider three scenarios: a relative infectivity during entire period is one (i.e., infectivity during the prodromal period and just prior to the rash is the same as during the first week of the rash), b the relative infectivity just prior to the rash is the same as during the first week of the rash, but during the prodromal period is 4% (as in Figure 3A ) of this value, and c the relative infectivity just prior to the rash is 20% of the infectivity during the first week of the rash, and during the prodromal period is 20% of this value. The figure shows that increased infectivity just prior to the rash leads to a larger epidemic (comparing b and c); in case b (high infectivity just prior to onset of rash), loss of containment occurs 36% of the time (but in none of the 100 realizations shown in case c (low infectivity prior to rash)). Scenario a (full infectivity during entire the prodromal period) showed loss of control in every realization. Increasing the ring vaccination capacity from 40 per day to 80 per day (results not shown) led to containment in all of the realizations with high infectivity just prior to the rash and low infectivity during the prodromal period (case b), but made no difference if the infectivity was as high during the prodromal period as during the rash (case a). While intuitively adding additional infectiousness must increase the number of secondary cases and make control more difficult, these results do illustrate that even a small amount of increased infectiousness prior to the rash (when diagnosis is more difficult) may substantially increase the difficulty of smallpox control.

Finally, in Figure 9 , we present scenarios in which each of four other parameters are modified from the baseline values of Figure 3A , assuming 40 contact tracings (ring vaccinations) are possible per day (line a in the figure) . Specifically, we assume that severe smallpox (hemorrhagic and flat) on average takes four times longer to diagnose and isolate than ordinary smallpox (case b), Table 4 : Containment of severe smallpox at different levels of contact finding. The first three columns are assumed levels for the probability of finding a household contact, the probability of finding a workplace/social (W/S) contact, and for the number of contact tracings/ring vaccinations possible per day; the last two columns express (as percentages) the resulting probability of containment given the assumed contact finding probabilities and contact tracing capacities; two containment probabilities are given: the containment probability when only contacts of cases are traced (first column, "Contacts"), and the containment probability when contacts of contacts of cases are traced in addition to the contacts of cases (second column, "Contacts of Contacts"). All other parameters are the same as in Figure 3A . that no one in the population has prior vaccination protection (from before the discontinuation of routine vaccination, case c), that 10% more smallpox is too mild to diagnose (but still contagious, case d) compared to baseline, and finally that the vaccine is completely ineffective (case e). Each of these scenarios will be discussed further below.

Scenario b was motivated by the possibility that individuals with severe forms of smallpox may be more difficult to diagnose, and thus remain infectious in the community longer (despite the much greater degree of illness of such patients), or that such patients may be more infectious. In this particular case, quadrupling the mean diagnosis time led to one additional replication out of 100 in which containment was not achieved (2/100, compared to the baseline of 1/100). However, we assumed that community awareness of smallpox leads to the same relative rate of increased diagnosis among severe cases as for ordinary cases, and that the most severe forms are relatively rare. In addition to the scenario shown in the figure, we also replicated the same 1000 "calibrated" simulations, assuming that in each case 40 contact tracings per day are possible and that the diagnosis time for severe cases was four times that of ordinary cases. Finally, we repeated each "calibrated" scenario 100 times assuming long diagnosis times for severe cases, and not making this assumption, and found that the difference in the decontainment fraction was not large (results not shown).

Scenario c illustrates that vaccination prior to the discontinuation of routine vaccination does play a role in smallpox control by ring vaccination; there were more decontainment scenarios (5/100) when no prior Transmission prior to the rash Figure 8 Transmission prior to the rash makes epidemic control more difficult. The figure shows a expanding smallpox epidemic assuming differing levels of infectivity prior to the rash. We assume all parameters are the same as in Figure 3A (and that the ring vaccination capacity is 40 per day). Infectivity prior to the rash is modeled as the relative infectivity during the short (1 day) period of oropharyngeal lesions just prior to the rash (compared to the infectivity during the first week of the rash), and as the relative infectivity during the prodromal period (relative to the period just prior to the rash). For scenario a, relative infectivity during the prodromal period and just prior to the rash is the same as during the first week of the rash, for scenario b, the relative infectivity just prior to the rash is the same as during the first week of the rash, but during the prodromal period is 4% (as in Figure 3A ) of this value, and for scenario c, the relative infectivity just prior to the rash is 20% of the infectivity during the first week of the rash, and during the prodromal period is 20% of this value (these two parameters are the same as in Figure 3A ). Additional scenarios, assuming 40 ring vaccinations or con-tact tracings possible per day, and that contacts of contacts are traced; all parameters are identical to those in Figure 3A unless otherwise indicated Figure 9 Additional scenarios, assuming 40 ring vaccinations or contact tracings possible per day, and that contacts of contacts are traced; all parameters are identical to those in Figure 3A unless otherwise indicated. The figure shows the average of 100 replications of five scenarios (Case a repeats the result from Figure 3A for reference); the numbers in parentheses in the legend are the corresponding fraction of the 100 scenarios for which decontainment occurred. For case b, we assumed that flat and hemorrhagic smallpox cases took four times as long on average to diagnose as ordinary cases; for case c., we assumed that no one in the population had prior protection (as opposed to 25% for Figure 3A) ; for case d, we assumed that an additional 10% of individuals (13% instead of 3%) would develop mild smallpox (with 75% developing ordinary smallpox instead of 85% as in Figure 3A ); and for case e, we assumed that the vaccine is completely ineffective and provides no protection against infection. protection exists in the population. The results suggest that prior vaccination aids in the control of smallpox, but that it is not strictly necessary for control (in this scenario, 95% of the replications exhibited containment). In Figure 3A , we assumed 25% of individuals had protection due to vaccination prior to the discontinuation of routine vaccination; in scenario c of Figure 9 , we assumed this fraction was zero.

Scenario d demonstrates that if 10% more smallpox infections (in absolute terms, i.e. 13% compared to 3% in Figure 3A ) lead to mild cases among individuals with no prior protection, the epidemic is more difficult to contain (13/100 replications showed loss of containment).

Finally, scenario e demonstrates that containment is still possible even when the vaccine is completely ineffective in everyone -because of case isolation and isolation of contacts (and of contacts of contacts). Here, with 40 contact tracings possible per day, 55% of the replications nevertheless exhibited containment even with a vaccine which offered no protection whatever. With 90 contact tracings possible per day, all replications exhibited containment even assuming no vaccine protection.

Although less efficient than ring vaccination in the sense that more vaccinations must be delivered to eliminate infection, comprehensive mass vaccination following the introduction of smallpox is sufficient to eliminate the infection. In Figure 10 , we show the probability of achieving containment (defined to be fewer than 500 total cases resulting from 10 index cases) for different levels of ring vaccination (0, 5, 10, and 20 vaccinations per day) and mass vaccination (0, 0.5%, 1%, and 2%; compare with the 10%-20% per day many jurisdictions in the United States are planning to vaccinate). Specifically, for each level of ring vaccination and mass vaccination, we used the same 1000 parameter sets used in Figure 5 , and performed 100 simulated epidemics for each parameter set. On the vertical axis, we plot the fraction of the 1000 scenarios for which each of the 100 simulated epidemics was contained. We further computed the fraction of scenarios for which none of the 100 simulated epidemics was contained; this is indicated by the colored segment in the small pie chart at each symbol. When the mass vaccination rate was 2% per day, the mean number of deaths (averaging over all scenarios and all simulations within each scenario) was 47.7, 33.7, 26.4, and 20.1 for a ring vaccination level of 0, 5, 10, and 20 per day (respectively) out of a population of 10000. Moreover, when we increased the mass vaccination level to 3%, an average of 28.9 deaths occurred when no ring vaccination was used, but this fell to 22.3 deaths when only 5 ring vaccinations per day were available (again assuming a population of 10000, and 10 index cases). With a mass vaccination level of 5% per day, an average of 18.8 deaths occurred without ring vaccination, and 15.8 deaths occurred when only 5 ring vaccinations per day were possible. (At a mass vaccination rate of 3% per day, containment as defined above was achieved in all 100 replications for 95% of the scenarios even without ring vaccination; at a mass vaccination rate of 5% per day, containment was achieved in all replications for all scenarios.) These results show that over a wide range of simulated epidemics, even seemingly small levels of ring vaccination (coupled with follow-up) may have a substantial effect in preventing epidemic spread and reducing deaths from smallpox, even during a mass vaccination campaign. Note that many jurisdictions in the United States are planning mass vaccination campaigns which could reach 10%-20% of the population per day, far greater than the mass vaccination levels examined here; it is interesting to note that mass vaccination cam- Figure 10 Mass and ring vaccination together. Low-level mass vaccination programs are improved substantially by the addition of ring vaccination. The shaded pie segments represent the fraction of 1000 scenarios for which containment (as defined in the text) was never realized; the vertical position of the pie chart represents the fraction of the 1000 "calibrated" scenarios for which containment was always achieved. As the fraction of the population mass vaccinated increases or the ring vaccination capacity increases, the probability of containment increases. paigns may be effective in preventing a widespread epidemic even at much lower levels than are being planned for. Where feasible, such rapid mass vaccination rapidly eliminates smallpox transmission in our model; vaccination of contacts is still beneficial, since we are assuming that earlier vaccination yields a greater probability of preventing or ameliorating infection (results not shown).

We constructed a simple network model of smallpox transmission, and addressed the question of what circumstances contribute to the success of a ring vaccination campaign designed to control smallpox. Our analysis focused on the use of contact tracing/ring vaccination to prevent a widespread epidemic following a deliberate release.

We conducted a sensitivity analysis based on particular, but reasonable, ranges for the unknown parameters. Our results are consistent with prior vaccination models in identifying prior vaccination and ring vaccination capacity as significant factors in determining the spread of smallpox. Unsurprisingly, we also find that household size and ring vaccination speed are particularly important parameters; these results are intuitively plausible. The contact finding probability did not appear important in this analysis only because a narrow range of values was chosen.

We illustrated smallpox control by presenting scenarios based on control of moderately severe smallpox epidemics. We find that swift, aggressive contact tracing and ring vaccination is is usually sufficient to bring the infection under control. Provided that there is sufficient capacity, vaccination of contacts of contacts is beneficial, and results in fewer infected individuals and more rapid elimination of infection; investigating contacts of contacts allows the chain of transmission to be outrun to some extent. When ring vaccination capacity is small, diversion of crucial resources away from contacts is harmful; contacts of contacts should only be traced and vaccinated provided that no resources are diverted away from contacts of cases. The increased surveillance (or isolation) of contacts, together with improved rates of diagnosis due to community awareness, play important roles in smallpox control; we note that in some cases, lowered diagnosis rates among severe cases contributed to a small extent to loss of epidemic control, and suggest that any public awareness campaign include information to help the public be more aware of the full spectrum of the clinical features of smallpox.

One limitation of our analysis is that we chose not to explicitly incorporate the specific epidemiology of health care workers (or mortuary workers), who are likely to be exposed to infected individuals during any smallpox epidemic (e.g. [17, 22] ), and who may then infect further members of the community [22] (as was also seen in the recent outbreak of SARS, e.g. [48] ). Transmission to health care workers may be considered to amplify the initial attack or to be simply accounted among the exposures we considered (and thus be approximated by the behavior of our model), since health care workers and their household contacts are in all likelihood traceable contacts, and ring vaccination/contact tracing would identify and halt these chains of transmission as in our model. The disruption of smallpox control and patient care that may occur is not accounted for in our analysis, however, causing our model in this sense to err on the side of optimism. The appropriateness of pre-event vaccination of health care workers or other first responders has been addressed by other analyses [12, 49] , and is beyond the scope of our model.

While we analyzed the effect of contact tracing, case and contact isolation, and ring vaccination (together with mass vaccination), in a real smallpox epidemic, in practice, control efforts are unlikely to be limited strictly to vaccinating contacts (and health care workers, as likely contacts) and isolating cases. Indeed, making vaccine available to individuals who believe they live near cases or to others on a voluntary basis occurred in smallpox control efforts in the past [22] . Vaccination of such individuals can only harm the disease control effort if it hinders or delays the diagnosis of cases or the investigation and vaccination of contacts; our results show that even relatively low levels of vaccination of the general population may have a beneficial effect in preventing the epidemic from escaping control.

More serious is the possibility that individuals who should be vaccinated or isolated would be missed; this could occur either because individuals or institutions did not cooperate with the disease control effort, or because the individuals simply could not be found. Our analysis suggests that ring vaccination need not be perfect to successfully contain the epidemic, and yet, under conditions where there is a high rate of infection among contacts, or a relatively high rate of casual transmission, high rates of contact finding (in excess of 90%), together with increased surveillance and contact isolation, are needed to contain the epidemic.

Finally, the vaccination of individuals at low risk of contracting smallpox will cause harm due to adverse events of the vaccine; in our model, the assumed death rate due to vaccination was small compared to the probability of death from smallpox, and played essentially no role in the analysis. In practice, individuals suspected to be at high-risk for vaccine complications, but at relatively low risk for contracting smallpox, might simply be isolated or closely monitored even during an outbreak; while the presence of individuals in the population at higher risk for vaccine complications would increase the death rate during an outbreak, such individuals are unlikely to impair the containment of the epidemic (the primary focus of this analysis).

Our results support ring vaccination against epidemics of smallpox (even assuming high rates of transmission to close contacts), but do note that stochastically, for severe (rapidly transmissible) smallpox, scenarios of loss of control are seen, with resulting widespread epidemics. In scenarios in which the transmission potential of smallpox is smaller, such loss-of-control scenarios occur less frequently (results not shown). Mass vaccination campaigns, when conducted quickly and with very high coverage, do not result in loss of control in our model. Nevertheless, fewer deaths due to smallpox result when ring vaccination is conducted along with mass vaccination.

Simulated smallpox epidemics with ring vaccination suggest that aggressive, fast ring vaccination can control epidemics of smallpox. To do so, however, smallpox must be identified quickly and contacts vaccinated promptly. We also identify public awareness of smallpox -leading to prompt identification of cases -as a major factor in smallpox control; in some simulations, it may play a role as significant as ring vaccination itself [15] . However, we also found that uncertainty in (1) transmission from mild cases, (2) the household size, and (3) casual transmission contributed to the overall uncertainty in the epidemic size. Other parameters to which the number of infections were highly sensitive were the prior vaccination fraction, parameters related to infectiousness, and parameters related to transmission prior to the rash.

Because our model combines network structure with response logistics, our results support and complement the results of other investigators. Our results support the notion that prior vaccine protection may play an important role in slowing the epidemic [11] , despite the possibility that some vaccinated individuals may develop mild cases which are harder to identify, but which nevertheless transmit disease. Likewise, our results provide support for the view that ring vaccination should play a central part in smallpox control. If initiated, ring vaccination should be conducted without delays in vaccination, should include contacts of contacts (whenever there is sufficient capacity to cover all contacts of cases), and should be accompanied by a vigorous campaign of public awareness which can facilitate more rapid identification and isolation of cases. We assumed that ring vaccination could be fast (little delay between identification of a case and vaccination of the contacts), effective (nearly all household contacts can be found, and most of workplace and social contacts), and available (there is sufficient capacity). To be effective, ring vaccination planning must yield a system capable of meeting these benchmarks; we should not only be able to assess the number of contact vaccinations that will be possible per day, but should have a plan in place to (1) identify contacts by working with individuals, employers, schools, community representatives, and authorities or businesses who may have access to information facilitating contact tracing, (2) rapidly investigate and vaccinate such individuals, perhaps using field teams managed by central dispatch. It is important to realize that for highrisk, transient, or unstably housed populations where reliable contact tracing is impossible, the conclusions of the model we present cannot be applied. It is important to note that while our model suggests that ring vaccination together with contact tracing and isolation is likely to be successful, we found that for some scenarios (where smallpox was more transmissible, or was relatively more transmissible before the rash), epidemic containment required not only ring vaccination, but increased public awareness, the isolation of contacts, and tracing of contacts of contacts. For scenarios in which the smallpox was less transmissible, epidemic containment was possible at lower contact finding probabilities. Thus, while our simulations suggest that contact tracing/ring vaccination need not be perfect to succeed, because of uncertainties in our knowledge of the behavior of bioterrorist smallpox, it is impossible to know in advance how good it will have to be. Thus, that high contact finding rates, mass public awareness leading to early identification of cases, isolation of contacts, and investigation of contacts of contacts should all be conducted with maximum effectiveness to reduce the probability of a widespread epidemic.

While the possibility of smallpox uncontrollable by ring vaccination has made mass vaccination preparations wise, and while mass vaccination may be unavoidable in the event of a deliberate release of smallpox, we believe that ring vaccination is essential in any case. This is not only because individuals recently exposed to smallpox may be protected if they are vaccinated promptly, but because each contact identified potentially lies in the immediate future of the transmission chain. From the standpoint of epidemic control, it is far more valuable to vaccinate individuals next in the transmission chain than to vaccinate other persons. Our results support the idea that ring vaccination/case isolation may in many, if not most cases, eliminate smallpox even without mass vaccination, but also support planning for mass vaccination (so that the vastly more costly and difficult policy of mass vaccination will be available in the event of an explosive epidemic). When faced with the unknown, multiple redundant prep-arations are appropriate; case investigation/isolation may control smallpox even if the vaccine does not work at all, but mass vaccination is useful in the event of an explosive epidemic for which case tracking becomes impossible. Protection of pulmonary epithelial cells from oxidative stress by hMYH adenine glycosylase BACKGROUND: Oxygen toxicity is a major cause of lung injury. The base excision repair pathway is one of the most important cellular protection mechanisms that responds to oxidative DNA damage. Lesion-specific DNA repair enzymes include hOgg1, hMYH, hNTH and hMTH. METHODS: The above lesion-specific DNA repair enzymes were expressed in human alveolar epithelial cells (A549) using the pSF91.1 retroviral vector. Cells were exposed to a 95% oxygen environment, ionizing radiation (IR), or H(2)O(2). Cell growth analysis was performed under non-toxic conditions. Western blot analysis was performed to verify over-expression and assess endogenous expression under toxic and non-toxic conditions. Statistical analysis was performed using the paired Student's t test with significance being accepted for p < 0.05. RESULTS: Cell killing assays demonstrated cells over-expressing hMYH had improved survival to both increased oxygen and IR. Cell growth analysis of A549 cells under non-toxic conditions revealed cells over-expressing hMYH also grow at a slower rate. Western blot analysis demonstrated over-expression of each individual gene and did not result in altered endogenous expression of the others. However, it was observed that O(2 )toxicity did lead to a reduced endogenous expression of hNTH in A549 cells. CONCLUSION: Increased expression of the DNA glycosylase repair enzyme hMYH in A549 cells exposed to O(2 )and IR leads to improvements in cell survival. DNA repair through the base excision repair pathway may provide an alternative way to offset the damaging effects of O(2 )and its metabolites. Oxidative stress leading to the overproduction of free radicals in the lungs is present in many clinical situations. Such clinical settings include acute respiratory distress syndrome (ARDS), infants of prematurity going on to develop bronchopulmonary dysplasia (BPD), pathogenesis of chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, ischemia-reperfusion injury, druginduced lung toxicity, cancer and aging [1] [2] [3] [4] . Although the use of oxygen may be clinically indicated in hypoxemic situations, one must consider the potential longterm toxic side effects. For example, we know that oxygen creates cellular damage by a variety of mechanisms. Normal cellular metabolism of oxygen involves the transfer of electrons from NADH to O 2 molecules to form water (H 2 O). At normal partial pressure, 95% of oxygen molecules (O 2 ) are reduced to H 2 O and 5% are partially reduced to toxic byproducts by normal metabolism in the mitochondria [5] . These metabolites include the superoxide anion (O 2 -), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals ( • OH) all of which make up what are known as Reactive Oxygen Species (ROS) [6] . Exposure to conditions of hyperoxia as well as ionizing radiation (IR) leads to increased amounts of these ROS and their damaging effects.

ROS are known to attack the lipids, proteins, and nucleic acids of cells and tissues [5, 7] . Lipids, including pulmonary surfactant, react with ROS to produce lipid peroxides, which cause increased membrane permeability, inactivation of surfactant, and inhibition of normal cellular enzyme processes. Proteins reacting with ROS result in decreased protein synthesis due to inhibition of ribosomal translation or destruction of formed proteins. This ultimately leads to inactivation of intracellular enzymes and transport proteins resulting in impaired cellular metabolism and accumulation of cellular waste products. Lastly, ROS cause damage to nucleic acids by leading to modified purine and pyrimidine bases, apurinic (AP) / apyrimidinic sites, and DNA protein cross-links which can lead to single strand breaks [8] .

Several defense mechanisms exist to combat the damaging effects of ROS. Intracellular enzymatic systems include superoxide dismutase which eliminates the superoxide anion, catalase which catalyzes the reduction of H 2 O 2 directly to H 2 O without the production of the hydroxyl radical, and glutathione peroxidase which directly reduces H 2 O 2 and lipid peroxides. Free radical scavengers, which stop free radical chain reactions by accepting electrons, include α-tocopheral (vitamin E), ascorbic acid (vitamin C), niacin (vitamin B), riboflavin (vitamin B 2 ), vitamin A, and ceruloplasmin [1, 2, 9] . These systems usually provide enough protection against oxygen metabolism under normal conditions, but may become depleted under conditions of increased oxidative stress [7, 10] .

The defense mechanism of interest in this paper involves the repair of oxidative damage through the human DNA base excision repair pathway (BER). BER is the most important cellular protection mechanism that removes Base excision repair pathways for Oxidative DNA damage oxidative DNA damage [11] . Damaged bases are excised and replaced in a multi-step process. Lesion-specific DNA glycosylase repair genes initiate this process. After removal of the damaged base, the resulting AP site is cleaved by APendonuclease generating a 3'OH and 5'deoxyribose phosphate (dRP). β-polymerase, which possesses dRPase activity, cleaves the dRP residue generating a nucleotide gap and then fills in this single nucleotide gap. The final nick is sealed by DNA ligase [12] [13] [14] ( Figure 1A ).

The oxidative repair genes that we have analyzed in this study include 8-oxoguanine DNA glycosylase (hOgg1), human Mut Y homologue (hMYH), human Mut T homologue (hMTH), and endonuclease III (hNTH) all of which are present in human cells and involved in the protection of DNA from oxidative damage. The repair enzyme hOgg1 is a purine oxidation glycosylase that recognizes and excise 8-oxoguanine lesions (GO) paired with cytosine. GO can pair with both cytosine and adenine during DNA replication [15] . If repair of C/GO does not occur, then G:C to T:A transversions may result [5, [15] [16] [17] . The repair enzyme hMYH is an 8-oxoguanine mismatch glycosylase that removes adenines misincorporated opposite 8-oxoG lesions that arise through DNA replication errors [5, [18] [19] [20] . The repair enzyme hMTH hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-dGTP, 8-oxo-GTP, 8-oxo-dATP, and 2-hydroxy-dATP, effectively removing them from the nucleotide pool and preventing their incorporation into DNA ( Figure 1B ) [21] . Lastly, the repair gene endonuclease III (hNTH) is a pyrimidine oxidation and hydration glycosylase that recognizes a wide range of damaged pyrimidines [22] . hNTH has also been shown to have a similar DNA glycosylase/AP lyase activity that can remove 8-oxoG from 8-oxoG/G, 8-oxoG/A, and 8-oxoG/C mispairs [23, 24] . Subsequent steps following hNTH are identical to those following hOgg1 ( Figure 1A ).

A previous study has shown that over-expression of the DNA repair gene hOgg1 leads to reduced hyperoxiainduced DNA damage in human alveolar epithelial cells [25] . The primary goal of our present study was to compare the protective effects of the four main lesion-specific DNA glycosylase repair genes by individually overexpressing each in lung cells and determining which of these provides the greatest degree of protection under conditions of increased oxidative stress.

The human alveolar epithelial cell line A549 (58 year old Caucasian male), was purchased from ATCC Cat No CCL-185. The cells were grown in DMEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT) and penicillin (100 U/ml)/ streptomycin (100 µg/ml) (Gibco, Grand Island, NY).

Passaging of cells was performed every 3-4 days with cells grown to 80% confluency in a 10 cm cell culture dish (Corning Incorporated, Corning, NY). Cells were kept at 37°C in a humidified, 5% CO2 incubator.

The retroviral vector pSF91.1, a gift from Dr. C. Baum from the University of Hamburg in Germany, was constructed with an internal ribosome entry site (IRES) upstream to the gene expressing enhanced green fluorescent protein (EGFP) as previously described [26] .

Four DNA repair genes were individually ligated into the retroviral vector pSF91.1.

hOgg1-6pcDNA3.1 was initially amplified by PCR by primers to introduce a kozak sequence at the 5' end [27] . Digestion of this product with EcoRI and SalI was performed and then hOgg1 was subcloned into digested plasmid vector pSF91.1, with T4 DNA ligase. DNA sequencing was performed to confirm integrity of the hOgg1 gene.

hMYH/PGEX4T-1 and hMTH/PGEX4T-1 hMYH was a gift from Dr. A. McCullough (University of Texas Medical School, Galveston, TX) and hMTH was cloned in Dr. Kelley's lab. Plasmid DNA was prepared as above by digestion with EcoRI and SalI and ligated into pSF91.1 as above and sequencing was performed to confirm integrity of the genes.

PGEX-6PI-hNTH1-wild type this gene was a gift from Dr. S. Mitra (University of Texas Medical School, Galveston, TX). Digestion with BamHI and SalI was performed and the hNTH1-wt fragment was ligated into the empty plasmid vector PUC18. The hNTH1-wt fragment was then excised with both sides flanked by EcoRI restriction sites and ligated into pSF91.1. Proper orientation of the gene was confirmed and sequencing was performed to determine the integrity of the gene. 2.5 × 10 5 A549 cells were suspended with the viral supernatant and plated in 1 well of a 6-well plate along with polybrene (Sigma, St. Louis, MO). This exposure was performed 6 hours per day for three days. At approximately five days from the beginning of the infection, the infected cells were analyzed using flow cytometry and sorted for EGFP expression.

Cell pellets of sorted cells were resuspended in NuPage buffer (Invitrogen, Carlsbad, CA) and protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA). 20 ug of protein were loaded into individual lanes of a NuPage Bis-Tris Gel (Invitrogen, Carlsbad, CA). The gel was then transferred to nitrocellulose paper (Osmonics Inc, Gloucester, MA). The membranes were then blocked with 1% blocking solution (Roche Diagnostics, Indianapolis, IN) for 1 hour at room temperature and then incubated overnight at 4°C with rabbit polyclonal antibodies to hOgg1 (Novus Biologicals, Littleton, CO), hMTH (Novus Biologicals, Littleton, CO), hMYH (Oncogene Research Products, Darmstadt, Germany) and hNTH (Proteintech Group Inc, Chicago, IL) all at a dilution of 1:1000 except hNTH which was diluted 1:2500. They were then washed 2 times with TBST and 2 times with 0.5% blocking solution, 10 minutes per wash. The membranes were incubated with anti-rabbit secondary antibodies at 1:1000 for 1 hour at room temperature. Lastly, the membranes were washed 4 times with TBST, 15 minutes per wash. The membranes were briefly soaked in BM chemiluminescence blotting substrate (Roche Diagnostics, Indianapolis, IN) and then exposed to high performance autoradiography film (Amersham Biosciences, Buckinghamshire, England). Kodak Digital Science 1D Image Analysis software was utilized to quantify the region of interest (ROI) band mass of individual bands on films where visualized differences were detected.

Sorted EGFP positive A549 cells infected with the above DNA repair genes were counted and seeded into 96-well plates at a density of 1000 cells/well, 6 wells per gene. Six hours after seeding, individual plates were placed into an oxygen chamber supplied by Dr. L. Haneline (Wells Center for Research, Indianapolis, IN) located in a 37°C incubator. The oxygen chamber was then infused with 95% O 2 and 5% CO 2 . Individual plates were removed after 12, 24, 48, and 72 hours of exposure. Control A549 cells were incubated in a normal 37°C humidified-5% CO 2 incubator. O 2 concentrations were monitored with a MAXO 2 analyzer (Maxtec, Salt Lake City, UT). Four days from the beginning of the exposure, cells were assessed for cell growth/survival using the sulforhodamine B assay (SRB assay).

The SRB assay (Sigma, St. Louis, MO), developed by the National Cancer Institute, provides a sensitive measure of drug-induced cytotoxicity through a colorimetric endpoint that is non-destructive, indefinitely stable, and visible to the naked eye. This assay was used to assess the cell growth/survival of over-expressed cells [28] . Cold 10% TCA was used to fix the cells to the plate. After incubation for 1 hour at 4°C, the individual wells were rinsed with water. After air-drying, SRB solution was added to each well and cells were allowed to stain for 20-30 minutes. 1% acetic acid wash was used to rinse off unincorporated dye. Incorporated dye was then solubilized in 100 µl per well of 10 mM Tris. Absorbance was measured by a tunable microplate reader (Molecular Devices, Sunnyvale, CA) at a wavelength of 565 nm. Background absorbance measured at 690 nm was subtracted from the measurements at 565 nm.

Sorted EGFP positive A549 cells were seeded into 96-well plates at a density of 1000 cells/well. Six hours after seeding, individual plates were then exposed to radiation at doses of 250, 500, 1000, and 1500 Rads or 0. well. All the plates were placed into a 37°C humidified-5% CO 2 incubator. Every 24 hours for 4 days, 1 plate was removed and the cells were fixed and analyzed by the SRB assay looking at cell growth under non-toxic conditions. Growth curves and exponential growth equations were determined to look at the doubling time (DT) of cells infected with each repair gene of interest compared to vector infected and uninfected wild type cells.

All drug exposure experiments were performed at least three times and individual drug doses included 6-8 wells for each group of infected cells. Analysis of cell growth and exponential growth equations were determined using Microsoft Excel. All experiments involving drug exposures were normalized to the zero dose. Data are expressed as means ± SE. The significance of differences were calculated using the paired Student's t test with significance being accepted for p < 0.05.

The DNA repair genes hOgg1, hMYH, hMTH, and hNTH were ligated into the retroviral vector pSF91.1 ( figure 2 ). This vector, derived from a murine stem cell virus backbone, along with each individual repair gene, was used for transfection of phoenix amphotropic cells. Viral supernatant was then collected and used to stably infect A549

Western analysis of A549 cells over-expressing individual repair genes and effect on endogenous glycosylase level contained the genes of interest integrated into their DNA (data not shown).

Western blot analysis was performed on sorted cells in order to verify over-expression of the four genes of interest. hOgg1, hMYH, hMTH, and hNTH were all detected at their correct position on western blots (data not shown).

Western analysis was also utilized to assess whether overexpression of each individual repair gene resulted in altered endogenous expression of the other repair genes under both non-toxic and toxic conditions (24 hrs of 95% O 2 and 1000 Rad). Cells over-expressing the repair genes hOgg1, hMYH, hMTH, and hNTH did not lead to altered expression of the other endogenous repair genes under the above conditions when compared to each other or pSF91.1 vector control cells ( Figure 3A ,3B,3C and 3D). hOgg1's endogenous expression was below the level of detection. The pattern of endogenous expression of hNTH was consistent for each condition when comparing cells over-expressing hOgg1, hMYH, hMTH, and pSF91.1. Reduced expression of hNTH after exposure to 95% O 2 was noted.

Lastly, we assessed endogenous expression of each individual repair gene in cells infected with pSF91.1 following non-toxic and toxic conditions (24 hrs of 95% O 2 and 1000 Rad) at 24 and 48 hrs after the onset of exposure. Endogenous hMYH and hMTH were expressed to the same degree. hOgg1's endogenous expression was below the level of detection using western analysis (results not shown). When analyzing endogenous hNTH expression, it was noted that hyperoxia at 24 hrs and 48 hrs resulted in reduced protein expression by 93% and 64% respectively. There also was a small increase in expression of hNTH noted after 1000 Rad one day post exposure that was back to baseline by two days post exposure. ROI band mass quantification demonstrated this finding ( Figure 4A and 4B). Two or more replicates were performed for each western analysis to determine consistency of the results.

A549 cells expressing hMYH demonstrated increased survival after exposure to conditions with elevated levels of oxygen compared to cells expressing only the pSF91.1 vector ( Figure 5A ). Results were highly significant at all time points except after 12 hours O 2 where it almost reached a highly significant value. The differences between pSF91.1 and hMYH varied from 12% after 12 hours O 2 exposure to 7% after 72 hours O 2 exposure. A549 cells expressing hMYH also demonstrated increased survival after exposure to all doses of radiation in comparison to pSF91.1 ( Figure 5B ). These results were also highly significant at all doses of radiation except at 250 Rads where it almost reached a highly significant value. The differences between pSF91.1 and hMYH varied from 12%-14% for all doses of radiation. Also noted in these experiments was that vector control cells demonstrated no Experiments looking at the effects of H 2 O 2 on cells expressing the repair genes did not demonstrate increased survival for any of these repair genes when compared to vector control cells ( Figure 5C ). This data demonstrates that over-expression of hMYH has the ability to improve cellular survival under conditions of hyperoxia and radiation but may not be able to overcome the toxicity of H 2 O 2 .

Cell growth under normal conditions was ascertained to determine if over-expression of any of the repair genes caused an alteration in the growth of cells in the absence of oxidative stress. Wild type A549 cells and cells expressing pSF91.1, hNTH, hOgg1, and hMTH appeared to grow at similar rates with doubling times within the same range. A549 cells expressing hMYH did show a slower growth rate that resulted in significant differences in cell number by day 3. The calculated doubling time for the cells over expressing hMYH is > 3 hrs longer than the cells with the other repair genes and vector alone ( Figure 6 ). This slowing of growth may allow for more time to repair Cell survival analysis following O 2 , IR, and H 2 O 2 treatments

Oxidative stress to the lung leads to cellular DNA damage as evidenced by the release of specific gene products known to regulate DNA base excision repair pathways such as p53 and p21 [29] [30] [31] . Alterations in pro-inflammatory mediators, transcription factors, and other related gene products are also observed [32] . This injury has been shown to be associated with features of both cellular necrosis and apoptosis [33] [34] [35] . The resultant cellular inflammation and death from oxidative stress has a dramatic impact on the outcome of patients in the clinical setting [7, 36] .

Most of our current clinical therapy towards oxidative stress in the lung involves both supportive measures and prevention. Research dealing with oxidative lung injury has focused mainly on enhancing antioxidant enzymatic processes and free radical scavengers [37] [38] [39] [40] . The ability to alter cellular survival by increasing specific DNA repair mechanisms may add another approach to the treatment of oxidant-mediated lung injury.

Many investigators have used hydrogen peroxide as a substitute for hyperoxia since it is known to be one of the metabolites produced by the metabolism of oxygen. ROS such as H 2 O 2 and those produced by hyperoxia clearly lead to DNA damage but questions exist as to whether H 2 O 2 leads to the same deleterious effects upon DNA as hyperoxia. Analysis of our growth curves after exposure to H 2 O 2 in comparison to hyperoxia and IR clearly indicates that cellular protection by oxidative DNA repair genes is specific to the agent used. Because no protection was observed with over-expression of any of the repair genes following exposure to H 2 O 2 , we speculate that the damage it causes is dissimilar. It may be that its damage not only involves oxidized bases, but may also include other forms of DNA, lipid, and protein damage that are not corrected by oxidative DNA repair genes. Alternatively, the amount and type of damage evoked by H 2 O 2 could be beyond that which can be corrected by over-expressing these repair genes.

Another form of stress known to induce damage through the formation of ROS is IR. Radiation induced free radical damage to DNA has substantial overlap with that of oxidative damage [41] [42] [43] . The protection provided by specific oxidative DNA repair genes under conditions of IR, was notable throughout our experiments only with the repair enzyme hMYH.

The primary agent utilized to induce the formation of ROS was an oxygen rich environment. The use of oxygen as a stressor leading to the formation of ROS, offers a distinct advantage over IR and H 2 O 2 by mimicking the clinical situation where constant exposure to hyperoxia leads to cumulative cellular damage which further compromises repair. We determined that survival of A549 cells was also enhanced to a small degree with increased expression of the repair enzyme hMYH. This was an unexpected finding as we anticipated the repair gene hOgg1 would demonstrate the greatest protection in response to oxidative stress based on previous studies, however these experiments utilized the colony forming assay (CFA) to detect improvements in survival [25] . Additionally, the CFA may provide different results compared to the SRB assay, which allows for growth analysis over a shorter window of time. Furthermore, their study did not look at the repair enzyme hMYH and its impact on survival. Another study has investigated the repair function of hMYH in MYH-deficient murine cells. It was demonstrated that transfection of the MYH-deficient cells with a wild-type MYH expression vector increased the efficiency of A:GO repair [44] .

An interesting observation noted while doing our experiments lead us to look at individual growth characteristics of cells over-expressing each of the oxidative repair enzymes. Cells over-expressing the repair enzyme hMYH clearly grow at a slower rate when compared with the other enzymes. The mechanism behind this is not understood at this point in time. The repair action of Cell growth curve and associated doubling times (DT) Figure 6 Cell growth curve and associated doubling times (DT hMYH is known to remove adenines misincorporated opposite 8-oxoG lesions. This lesion occurs when a C/GO lesion is allowed to replicate before being corrected by hOgg1. Repair by hMYH is not a final corrective measure. The product of hMYH activity is the lesion C/GO, which allows hOgg1 to have another opportunity to remove 8-oxoG opposite cytosine. We know that A549 cells possess the hOgg1 gene based on a previous study demonstrating the presence of this gene after amplification by genomic PCR [45] . We also have demonstrated endogenous activity of hOgg1 in A549 cells by using an 8-oxoguanine bioactivity assay. Therefore, our explanation of these results is that the slowed growth created by hMYH may provide a wider window of opportunity for the repair process to take place, which ultimately grants endogenous hOgg1 another opportunity to remove the 8-oxoG lesion created by oxidative stress.

As noted in the methods section, the SRB assay provides a sensitive measure of drug-induced cytotoxicity that is used to assess cell proliferation/survival. The reduced cell proliferation of A549 cells over-expressing hMYH under nontoxic conditions may likely underestimate the magnitude of the protective effect of this particular repair enzyme. This may in fact make the results even more significant.

Recent studies have discovered hereditary variations of the glycosylase hMYH that may predispose to familial colorectal cancer [46, 47] . Others have looked for hMYH variants in lung cancer patients and have not identified any clear pathogenic biallelic hMYH mutations or an overrepresentation of hMYH polymorphisms [47] . The A549 cell line has not demonstrated somatic mutations in hMYH, but a single nucleotide polymorphism (SNPs) has been noted [45] . The impact on function by this SNP is unknown. It would appear that the function of hMYH is very important in preventing somatic mutations leading to cancer in the gastrointestinal tract. Although studies to date have not demonstrated this same relationship with lung cancer, we do know that the lungs are subjected to large quantities of ROS under certain conditions as discussed earlier. The formation of mutations from oxidative stress does have other deleterious effects on cells including cellular death by necrosis and apoptosis. Tissue viability is dependent upon mutation correction and replication of the surviving cells to replace those that have died. The ability to enhance cellular survival, after specific oxidative exposures, is evident after increased production of the hMYH repair gene in these experiments.

We additionally wanted to determine the level of endogenous expression of the glycosylase repair genes in the pulmonary epithelial A549 cell line. Others have demonstrated how different stressors lead to alterations in the endogenous production of specific repair genes. For example, it has been shown that endogenous gene expression of hOgg1 was elevated following exposure to crocidolite asbestos which is known to cause an increase in 8-oxoG levels [48] . It has also previously been reported that treatment of A549 cells with sodium dichromate, a prooxidant, leads to a reduction of hOgg1 protein expression that was not observed with H 2 O 2 [49] . One additional study demonstrated a dose dependent down regulation of hOgg1 protein expression in rat lung after exposure to cadmium, a known carcinogen associated with the formation of intracellular ROS [50] . In our experiments we were able to demonstrate that both hyperoxia and IR do not appear to impact the endogenous expression of hOgg1, hMYH, and hMTH at 24 and 48 hours following exposure. It was noted that endogenous hNTH was reduced after hyperoxia at 24 and 48 hours after the onset of exposure. One would speculate that this reduction in endogenous hNTH secondary to hyperoxia is related to either decreased production or increased destruction in response to O 2 exposure. Over-expression of this repair enzyme did not result in improvements in survival after O 2 exposure based on our experiments. It may be that endogenous levels are adequate to correct this specific mutational burden for these experiments.

Furthermore, no previous studies have determined how cells over-expressing specific repair genes may impact endogenous expression of the other oxidative BER genes under both normal and oxidative stress conditions. We were also able to demonstrate that endogenous expression of glycosylase repair genes were not altered under these conditions secondary to the over-expression of any of these genes. This is an important finding for interpretation of survival data; protection of cells is due to the overexpression of the specific gene and not due to enhancement of other endogenous repair enzyme levels, at least for the genes studied under these conditions. Some limitations may exist in using a lung carcinoma cellline, which likely differs both in proliferative properties as well as in response to oxidative stress in comparison to primary epithelial cells. The enhanced cell growth observed with cell lines may be more reflective of undifferentiated alveolar type II cells which are likely to replace terminally differentiated alveolar type I cells after injury/ death due to oxidative stress. This may not be a true reflection of growth under non-toxic conditions when very little cell division is occurring. This is an inherent problem observed when comparing cell lines with primary cells and results need to be interpreted in a way that considers this.

It is difficult to know how this will translate to pulmonary epithelial cells in vivo at this stage. It certainly would appear that the protection observed is modest in degree in this pulmonary epithelial cell line. Further experiments assessing the function of the repair enzyme hMYH in this model will be important to perform in order to delineate the findings of slowed growth under normal conditions and improved survivability under conditions of O 2 and IR. More research looking at the potential for combination therapy, including DNA repair mechanisms in conjunction with other antioxidant defense mechanisms may be another approach to enhancing cell survival, which may lead to better clinical outcomes. Alternatively, cell survival may not be the most important end point for hyperoxia studies. Given that 8-oxoG, if left unrepaired, leads to G:C to T:A transversions, there may be an increase in mutational burden by these cells that isn't reflected in cell survival. Further experiments studying the impact on mutation production is underway. Ultimately, experiments need to be done in animal models to determine the translation to in vivo pulmonary cells.

In summary, we have demonstrated that over-expression of the DNA glycosylase repair enzyme hMYH may enhance survival of a pulmonary epithelial cell line after exposure to conditions of IR and hyperoxia. We have also demonstrated that over-expression of hMYH leads to a slowing of growth of A549 cells under non-toxic conditions, which may in part play a role in this enhancement of survival by providing a wider window of opportunity for repair of oxidized lesions to occur. Lastly, we demonstrated that over-expression does not lead to altered endogenous expression of these repair genes. As the understanding of DNA repair mechanisms continues to grow and the evolution of gene therapy takes place, more treatment options may be available in the clinical setting to help with many disease processes including the damaging effects of oxygen and its metabolites. Bioinformatic mapping of AlkB homology domains in viruses BACKGROUND: AlkB-like proteins are members of the 2-oxoglutarate- and Fe(II)-dependent oxygenase superfamily. In Escherichia coli the protein protects RNA and DNA against damage from methylating agents. 1-methyladenine and 3-methylcytosine are repaired by oxidative demethylation and direct reversal of the methylated base back to its unmethylated form. Genes for AlkB homologues are widespread in nature, and Eukaryotes often have several genes coding for AlkB-like proteins. Similar domains have also been observed in certain plant viruses. The function of the viral domain is unknown, but it has been suggested that it may be involved in protecting the virus against the post-transcriptional gene silencing (PTGS) system found in plants. We wanted to do a phylogenomic mapping of viral AlkB-like domains as a basis for analysing functional aspects of these domains, because this could have some relevance for understanding possible alternative roles of AlkB homologues e.g. in Eukaryotes. RESULTS: Profile-based searches of protein sequence libraries showed that AlkB-like domains are found in at least 22 different single-stranded RNA positive-strand plant viruses, but mainly in a subgroup of the Flexiviridae family. Sequence analysis indicated that the AlkB domains probably are functionally conserved, and that they most likely have been integrated relatively recently into several viral genomes at geographically distinct locations. This pattern seems to be more consistent with increased environmental pressure, e.g. from methylating pesticides, than with interaction with the PTGS system. CONCLUSIONS: The AlkB domain found in viral genomes is most likely a conventional DNA/RNA repair domain that protects the viral RNA genome against methylating compounds from the environment. The purpose of this study has been to identify domains with homology to AlkB in viral genomes, in order to get a better understanding of distribution and possible function of such domains. The AlkB protein of E. coli, and probably most of its homologues, is involved in repair of alkylation damage in DNA and RNA. It repairs 1-methyl-adenine and 3-methylcytosine by oxidative demethylation and direct reversal of the methylated base back to its unmethylated form. Recently the protein was identified as a member of the 2-oxoglutarate (2OG)-and Fe(II)dependent oxygenase superfamily [1] [2] [3] . The catalytic reaction requires molecular oxygen, Fe 2+ and 2-oxoglutar-ate, which is subsequently converted into succinate, CO 2 and formaldehyde [4] .

The 2OG-FeII oxygenase superfamily is widespread in Eukaryotes and bacteria [1] , and is currently the largest known family of oxidising enzymes without a heme group [5] . The 3D structure of several of these oxygenases is known, and they share a common fold with a structurally conserved jelly roll β-sheet core with flanking α-helices. Very few residues are totally conserved across these structures, basically just the residues involved in coordination of the Fe(II) ion and the 2-oxoglutarate.

AlkB-like genes are widespread in most types of organisms except Archaea. However, whereas bacteria normally have just one or at most two AlkB homologues [6] , multicellular Eukaryotes tend to have several homologues. In the human genome at least 8 different AlkB homologues (ABHs) have been identified [7] . These homologues seem to have slightly different properties with respect to substrate preference and subcellular localisation, and this may be a reason for the proliferation of ABHs e.g. in humans. However, a detailed functional mapping of all ABHs has not yet been carried out.

A sequence alignment of known ABHs identifies very few residues as totally conserved, basically just a HxD motif, a H and a RxxxxxR motif. These residues are also conserved in the more general 2OG-FeII oxygenase superfamily as described above, except for the final R. The first three residues (HxD and H) are involved in Fe(II)-coordination, whereas the first R is involved in 2OG-coordination. The final R is most likely involved in AlkB-specific substrate binding.

In addition to DNA repair, it has been shown that E. coli AlkB and the human AlkB homologue hABH3 may be involved in RNA repair. When expressed in E. coli both AlkB and hABH3 reactivate methylated RNA bacteriophage MS2 in vivo. This illustrates that direct repair may be an important mechanism for maintenance of RNA in living cells [4] . RNA repair proceeds by the same mechanism as DNA repair. Repair of damaged RNA was previously considered very unlikely, due to the natural redundancy of RNAs in a cell [8] . However, RNA is essential for cell function: unrepaired RNA can lead to miscoded or truncated proteins, and alkylated RNA could signal cell cycle checkpointing or apoptosis [9] . Consequently the occurrence of RNA repair does not come as a great surprise. The mechanism of direct reversal of methylation as used by AlkB homologues is particularly important for RNA repair, as it means that single-stranded regions may be repaired without introducing strand breaks. Repair of alkylation damage in DNA and RNA has recently been reviewed [10] .

AlkB homologues have also been found in plant viruses. It has been suggested that methylation may be used in host-mediated inactivation of viral RNAs, and that AlkB homologues in some plant viruses may be used to counteract such defence mechanisms [1] . However, no detailed study of this has been published.

The research project reported here has focused on a better understanding of the distribution and potential function of putative AlkB homology domains by using in silico mapping of viruses in which such domains have been found, as well as related viruses.

The general mapping strategy of the project was to identify viral genomes with AlkB homology domains, identify common features of these genomes, and subsequently find additional genomes with similar features, but without AlkB homology domains. This data set could then be used to analyse the properties and distribution of AlkBlike domains in viruses, as a basis for generating hypotheses about the evolution and function of these domains.

The PSI-Blast search for viruses in the NCBI nr protein sequence database was initiated with ALKB_ECOLI (NCBI gi113638), restricted to residues 110 to 210 and using the default inclusion threshold of 0.005 on E-values. The [11] .

In all of these viruses the AlkB domain is a part of the replicase polyprotein, which normally consists of a viral Other Pfam domains -Peptidase_C21, C23, C33, C34, C35 and C41, A1pp and OTU -were also identified in subsets of sequences. A1pp is a member of the Appr-1-p processing enzyme family, and the domain is found in a number of otherwise unrelated proteins, including non-structural proteins of several types of ssRNA viruses. OTU is a mem-ber of a family of cysteine proteases that are homologous to the ovarian tumour (otu) gene in Drosophila. Members of this family are found in Eukaryotes, viruses and pathogenic bacteria.

The MT, HEL and RdRp domains identified by Pfam as described above were extracted from the library sequences, aligned by ClustalX, and combined into a new alignment consisting of only these domain regions. This turned out to be necessary in order to get robust alignments. The intervening regions between the conserved domains are extremely variable in these sequences, and this tended to confuse alignment programs in the sense that conserved regions were not correctly aligned. The combined sequence alignment of domains from Closteroviridae, Flexiviridae and Tymoviridae was then used as input for building a phylogenetic tree with MEGA2. The final tree is shown in Figure 2 , with polyproteins containing AlkB-like domains indicated.

A second alignment was generated from all sequences with AlkB-like domains, using only the regions corresponding to MT, AlkB, HEL and RdRp Pfam domains. The domains were aligned individually, and the combined alignment was used as input for MEGA2. However, this data set did not give a reliable phylogeny (data not shown), and the separate domains of this alignment were therefore analysed individually and compared. This analysis is summarised in Tymoviridae measures (including SJA) for comparison of random trees [12] . The SJA values shown in Table 2 for comparisons between MT, HEL and RdRp NJ trees were 14.2 -17.1 standard deviations from the expectation value of 0.665 for a tree with 22 nodes, whereas the corresponding values for the AlkB NJ tree were 4.4 -5.4 standard deviations from the expectation value. Similar ranges were observed for the ML trees as well as for alternative distance measures, e.g. the Symmetric Difference (SD) measure (data not shown). Although this means that the SJA value for comparing AlkB trees to MT, HEL and RdRp trees were significantly better than for random trees, it also shows that the MT, HEL and RdRp trees were clearly more similar to each other than to the AlkB tree.

The alignment of the AlkB domain seemed to be of comparable quality to the other alignments. In fact the AlkB domain had the highest average pairwise sequence identity, as seen in Table 2 (see Figure 3 for the actual alignment). In other words, these AlkB domains were as similar to each other as the other three domains with respect to sequence identity, but they did not represent a consistent evolutionary history when compared to the other domains of this polyprotein. This may indicate that the AlkB domains have evolved separately from the other domains, and possibly as several independent instances.

The degree of co-evolution was analysed by computing pairwise distances between sequence regions in the alignment of MT, AlkB, HEL and RdRp domains described above. In Figure 4 selected results are shown as scatter plots, where the Blosum 50 score value between e.g. the MT domains in a pair of sequences is plotted against the score value for AlkB domains in the same pair of sequences. Plots for the MT, HEL and RdRp domains show that they are strongly correlated for MT vs. RdRp (r 2 = 0.95), MT vs. HEL (r 2 = 0.87) and HEL vs. RdRp (r 2 = 0.81). The plot of the AlkB domain vs. these three domains for the same set of sequences shows a very low degree of correlation for AlkB vs. RdRp (r 2 = 0.10), AlkB vs. MT (r 2 = 0.12) and AlkB vs. HEL (r 2 = 0.16).